Bacteriophage lysin and antibiotic combinations against gram positive bacteria

ABSTRACT

The present invention provides compositions and methods for prevention, amelioration and treatment of gram positive bacteria, particularly  Staphylococcal  bacteria, with combinations of lysin, particularly Streptococcal lysin, particularly the lysin PlySs2, and one or more antibiotic, including daptomycin, vancomycin, oxacillin, linezolid, or related antibiotic(s).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a § 371 of PCT Application No.PCT/US2013/040329 filed May 9, 2013, which in turn, claims priority fromU.S. Provisional Application Ser. No. 61/644,944 filed May 9, 2012 andU.S. Provisional Application Ser. No. 61/737,239 filed Dec. 14, 2012,the entire disclosures of the applications are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 3, 2020, isnamed 0341.0007_ST25.txt and is 8,031 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to prevention, amelioration andtreatment of gram positive bacteria, including Staphylococcal bacteria,with combinations of lysin, particularly Streptococcal lysin,particularly the lysin PlySs2, and one or more antibiotic.

BACKGROUND OF THE INVENTION

The development of drug resistant bacteria is a major problem inmedicine as more antibiotics are used for a wide variety of illnessesand other conditions. The use of more antibiotics and the number ofbacteria showing resistance has prompted longer treatment times.Furthermore, broad, non-specific antibiotics, some of which havedetrimental effects on the patient, are now being used more frequently.A related problem with this increased use is that many antibiotics donot penetrate mucus linings easily.

Gram-positive bacteria are surrounded by a cell wall containingpolypeptides and polysaccharide. Gram-positive bacteria include but arenot limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus,Staphylococcus, Streptococcus, Enterococcus, Mycobacterium,Corynebacterium, and Clostridium. Medically relevant species includeStreptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus,and Enterococcus faecalis. Bacillus species, which are spore-forming,cause anthrax and gastroenteritis. Spore-forming Clostridium species areresponsible for botulism, tetanus, gas gangrene and pseudomembranouscolitis. Corynebacterium species cause diphtheria, and Listeria speciescause meningitis.

Novel antimicrobial therapy approaches include enzyme-based antibiotics(“enzybiotics”) such as bacteriophage lysins. Phages use these lysins todigest the cell wall of their bacterial hosts, releasing viral progenythrough hypotonic lysis. A similar outcome results when purified,recombinant lysins are added externally to Gram-positive bacteria. Thehigh lethal activity of lysins against gram-positive pathogens makesthem attractive candidates for development as therapeutics (Fischetti,V. A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al(2001) Proc Natl Acad Sci USA 98:4107-4112). Bacteriophage lysins wereinitially proposed for eradicating the nasopharyngeal carriage ofpathogenic streptococci (Loeffler, J. M. et al (2001) Science 294:2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112).Lysins are part of the lytic mechanism used by double stranded DNA(dsDNA) phage to coordinate host lysis with completion of viral assembly(Wang, I. N. et al (2000) Annu Rev Microbiol 54:799-825). Lysins arepeptidoglycan hydrolases that break bonds in the bacterial wall, rapidlyhydrolyzing covalent bonds essential for peptidoglycan integrity,causing bacterial lysis and concomitant progeny phage release.

Lysin family members exhibit a modular design in which a catalyticdomain is fused to a specificity or binding domain (Lopez, R. et al(1997) Microb Drug Resist 3:199-211). Lysins can be cloned from viralprophage sequences within bacterial genomes and used for treatment(Beres, S. B. et al (2007) PLoS ONE 2(8):1-14). When added externally,lysins are able to access the bonds of a Gram-positive cell wall(Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400).Bacteriophage lytic enzymes have been established as useful in theassessment and specific treatment of various types of infection insubjects through various routes of administration. For example, U.S.Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection ofGroup A streptococci in clinical specimens, through the enzymaticdigestion by a semi-purified Group C streptococcal phage associatedlysin enzyme. This enzyme work became the basis of additional research,leading to methods of treating diseases. Fischetti and Loomis patents(U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use ofa lysin enzyme produced by group C streptococcal bacteria infected witha C1 bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis)discloses a composition for dermatological infections by the use of alytic enzyme in a carrier suitable for topical application to dermaltissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses amethod for treatment of bacterial infections of the digestive tractwhich comprises administering a lytic enzyme specific for the infectingbacteria. The carrier for delivering at least one lytic enzyme to thedigestive tract is selected from the group consisting of suppositoryenemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945(Fischetti and Loomis) discloses a method and composition for thetreatment of bacterial infections by the parenteral introduction(intramuscularly, subcutaneously, or intravenously) of at least onelytic enzyme produced by a bacteria infected with a bacteriophagespecific for that bacteria and an appropriate carrier for delivering thelytic enzyme into a patient.

Phage associated lytic enzymes have been identified and cloned fromvarious bacteriophages, each shown to be effective in killing specificbacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCTApplication WO2008/018854 provides distinct phage-associated lyticenzymes useful as antibacterial agents for treatment or reduction ofBacillus anthraces infections. U.S. Pat. No. 7,569,223 describes lyticenzymes for Streptococcus pneumoniae. Lysin useful for Enterococcus (E.faecalis and E. faecium, including vancomycin resistant strains) aredescribed in U.S. Pat. No. 7,582,291. U.S. 2008/0221035 describes mutantPly GBS lysins highly effective in killing Group B streptococci. Achimeric lysin denoted ClyS, with activity against Staphylococcibacteria, including Staphylococcus aureus, is detailed in WO2010/002959.

Based on their rapid, potent, and specific cell wall-degradation andbactericidal properties, lysins have been suggested as antimicrobialtherapeutics to combat Gram-positive pathogens by attacking the exposedpeptidoglycan cell walls from outside the cell (Fenton, M et al (2010)Bioengineered Bugs 1:9-16; Nelson, D et al (2001) Proc Natl Acad Sci USA98:4107-4112). Efficacies of various lysins as a single agents have beendemonstrated in rodent models of pharyngitis (Nelson, D et al (2001)Proc Natl Acad Sci USA 98:4107-4112), pneumonia (Witzenrath, M et al(2009) Crit Care Med 37:642-649), otitis media (McCullers, J. A. et al(2007) PLOS pathogens 3:0001-0003), abscesses (Pastagia, M et alAntimicrobial agents and chemotherapy 55:738-744) bacteremia (Loeffler,J. M. et al (2003) Infection and Immunity 71:6199-6204), endocarditis(Entenza, J. M. et al (2005) Antimicrobial agents and chemotherapy49:4789-4792), and meningitis (Grandgirard, D et al (2008) J Infect Dis197:1519-1522). In addition, lysins are generally specific for theirbacterial host species and do not lyse non-target organisms, includinghuman commensal bacteria which may be beneficial to gastrointestinalhomeostasis (Blaser, M. (2011) Nature 476:393-394; Willing, B. P. et al(2011) Nature reviews. Microbiology 9:233-243)

Antibiotics in clinical practice include several which commonly affectcell wall peptidoglycan biosynthesis in gram positive bacteria. Theseinclude glycopeptides, which as a class inhibit peptidoglycan synthesisby preventing the incorporation of N-acetylmuramic acid (NAM) andN-acetylglucosamine (NAG) peptide subunits into the peptidoglycanmatrix. Available glycopeptides include vancomycin and teicoplanin, withvancomycin a primary drug of choice and clinical application inbacteremia, particularly Staphylococcal infections. Penicillins act byinhibiting the formation of peptidoglycan cross-links. Commonpenicillins include oxacillin, ampicillin and cloxacillin. Linezolid(Zyvox) is a protein synthesis inhibitor and in a class ofantibacterials called oxazolidinones (Ford C W et al (1996) AntimicrobAgents Chemoth 40(6):1508-1513; Swaney S M et al (1998) AntimicrobAgents Chemoth 42(12):3251-3255; U.S. Pat. No. 6,444,813).

Daptomycin (Cubicin), also denoted LY 146032, is a lipopeptideantibacterial agent consisting of a 13-member amino acid peptide linkedto a 10-carbon lipophilic tail (Miao V et al (2005) Microbiology151(Pt5):1507-1523; Steenbergen J N et al (2005) J Antimicrob Chemother55(3):283-288; and described in U.S. Pat. No. 5,912,226). This structureresults in a novel mechanism of action, the disruption of the bacterialmembrane through the formation of transmembrane channels, which causeleakage of intracellular ions leading to depolarizing the cellularmembrane and inhibition of macromolecular synthesis. Daptomycin'sspectrum of activity is limited to Gram-positive organisms, including anumber of highly resistant species (methicillin-resistant S. aureus(MRSA), vancomycin intermediate-sensitive S. aureus (VISA),vancomycin-resistant S. aureus (VRSA), vancomycin-resistant Enterococcus(VRE)). In studies it appears to be more rapidly bactericidal thanvancomycin. Its approved dosing regimen is 4 mg/kg IV once daily. Doseadjustment is necessary in renal dysfunction. Daptomycin's primarytoxicity is reversible dose-related myalgias and weakness. Daptomycinhas been approved for the treatment of complicated skin and soft tissueinfections caused by gram positive bacteria, Staphylococcus aureusbacteremia and right-sided S. aureus endocarditis. Trials assessingdaptomycin's efficacy in treating complicated urinary tract infectionsand endocarditis/bacteremia are ongoing. Its approved dosing regimen is4 mg/kg IV once daily. Dose adjustment is necessary in renaldysfunction. Daptomycin's primary toxicity is reversible dose-relatedmyalgias and weakness. Resistance to daptomycin has been encounteredboth in vitro and in vivo after exposure to daptomycin. The mechanism(s)of resistance are not fully defined but likely relate to alterations ofthe cellular membrane. Multiple passages of Staphylococci andEnterococci in subinhibitory drug concentrations resulted in MICincreases in a stepwise fashion. Daptomycin binds avidly to pulmonarysurfactant and cannot be effectively used in treatment of pneumonia(Baltz R H (2009) Curr Opin Chem Biol 13(2):144-151).

The broad spectrum antibiotics in clinical use for treatment of grampositive infections, particularly including critical care antibioticssuch as vancomycin, are limited in use and application by their sideeffects of gastrointestinal upset and diarrhea and the development ofresistance, particularly in connection with continued or long-term use.

It is evident from the deficiencies and problems associated with currenttraditional antibacterial agents that there still exists a need in theart for additional specific bacterial agents, combinations andtherapeutic modalities, particularly without high risks of acquiredresistance. Accordingly, there is a commercial need for newantibacterial approaches, especially those that operate via newmodalities or provide new combinations to effectively kill pathogenicbacteria.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

The present application relates to combinations of bacteriophagelysin(s) with antibiotic for rapid and effective killing of grampositive bacteria. In accordance with the invention, the lysin PlySs2,which demonstrates broad killing activity against multiple bacteria,particularly gram-positive bacteria, including Staphylococcus andStreptococcus bacterial strains, provides remarkable synergy incombination with antibiotic(s) and can significantly reduce theeffective MIC doses required for antibiotic(s).

The lysin may be combined with broad spectrum gram positiveantibiotic(s), including one or more of vancomycin, daptomycin,linezolid or oxacillin, including related or similar antibiotics. In aparticular aspect, PlySs2 lysin is combined with daptomycin to providesynergistic killing activity against gram-positive bacteria, includingStaphylococci, particularly including MRSA. In a particular aspect,PlySs2 lysin is combined with vancomycin to provide synergistic killingactivity against Streptococci, including MRSA. In a particular aspect,PlySs2 lysin is combined with linezolid to provide synergistic killingactivity against Streptococci, including MRSA. In an aspect of theinvention, combination with PlySs2 lysin significantly reduces the doseof antibiotic required to kill a gram positive bacteria, such as S.aureus.

In accordance with the invention, combinations of PlySs2 lysin andantibiotic, including antibiotic of distinct type or class, particularlyincluding daptomycin, vancomycin, linezolid or oxacillin are effectiveto kill gram positive bacteria, including S. aureus, at lower doses orwith lower MIC than either alone. In an aspect of the invention, lowerdose formulations of lysin and of antibiotic, including suitable foradministration in combination or separately simultaneously or in series,are provided wherein the dose for effective killing or decolonization ofa gram positive infection are lower than the dose required if either areprovided alone. In particular, low dose formulations of antibiotic areprovided for administration in combination with lysin, particularlyPlySs2 lysin, administered simultaneously or in series, wherein the dosefor effective killing or decolonization of a gram positive infection ofthe antibiotic are lower in combination with the lysin than the doserequired if antibiotic is provided or administered alone.

In an aspect of the invention, lysins effective against Staphylococciare combined with one or more of daptomycin, vancomycin, linezolid oroxacillin, or related antibiotic compounds, to kill gram positivebacteria, including S. aureus, at lower doses or with lower MIC thaneither alone. In an aspect of the invention, lysins effective againstStaphylococci are combined with daptomycin, or related antibioticcompounds, to kill gram positive bacteria, including S. aureus, at lowerdoses or with lower MIC than either alone. In an aspect of theinvention, lysins effective against Staphylococci are combined with oneor more of vancomycin, or related antibiotic compounds, to kill grampositive bacteria, including S. aureus, at lower doses or with lower MICthan either alone. In a particular aspect the antibiotic is combinedwith PlySs2 lysin or a variant thereof. In an aspect of the invention,the combination of lysin with daptomycin circumvents the effect ofsurfactant to reduce daptomycin activity. In combination with lysin,such as PlySs2 lysin, daptomycin is rendered effective in killing S.aureus and in treating or ameliorating bacteremia in an animal. Thus, inan aspect of the invention, a method is provided for decolonization,inhibition or treatment of a S. aureus infection in an animal comprisingadministering to an animal a composition comprising or a combination ofPlySs2 lysin and daptomycin.

In accordance with the present invention, compositions and methodscomprising PlySs2 and one or more antibiotic are provided for theprevention, disruption and treatment of bacterial infection orcolonization. In its broadest aspect, the present invention provides useand application of a lysin having broad killing activity againstmultiple bacteria, particularly gram-positive bacteria, includingStaphylococcus, Streptococcus, Enterococcus and Listeria bacterialstrains, in combination with antibiotic, particularly in combinationwith daptomycin, vancomycin, linezolid or oxacillin, or a relatedantibiotic, for the prevention, amelioration or treatment of grampositive bacteria or gram positive bacterial infections. The inventionthus contemplates treatment, decolonization, and/or decontamination ofbacteria by administration of or contact with a combination of PlySs2lysin and one or more antibiotic wherein one or more gram positivebacteria, particularly one or more of Staphylococcus, Streptococcus,Enterococcus and Listeria bacteria, is suspected or present. In one suchaspect, PlySs2 lysin is combined with daptomycin. In a further aspect,PlySs2 lysin is combined with vancomycin. In another aspect, PlySs2lysin is combined with linezolid. In an additional aspect, PlySs2 lysinis combined with oxacillin. In each instance the antibiotic includes orencompasses related antibiotics, including those of the same class orfamily or with similar or related structures.

In accordance with the present invention, bacteriophage lysin derivedfrom Streptococcus suis bacteria are utilized in the methods andcompositions of the invention. The lysin polypeptide(s) of use in thepresent invention, particularly PlySs2 lysin as provided herein and inFIG. 29 (SEQ ID NO: 1), are unique in demonstrating broad killingactivity against multiple bacteria, particularly gram-positive bacteria,including Staphylococcus, Streptococcus, Enterococcus and Listeriabacterial strains. In one such aspect, the PlySs2 lysin is capable ofkilling Staphylococcus aureus strains and bacteria in combination withantibiotic, particularly in combination with daptomycin, vancomycin,oxacillin or linezolid, as demonstrated herein. PlySs2 is effectiveagainst antibiotic-resistant Staphylococcus aureus such asmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistantStaphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus(DRSA) and linezolid-resistant Staphylococcus aureus (LRSA). PlySs2 iseffective against vancomycin intermediate-sensitivity Staphylococcusaureus (VISA).

In an aspect of the invention, a method is provided of killinggram-positive bacteria comprising the step of contacting the bacteriawith a combination of PlySs2 lysin and one or more antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, theisolated lysin polypeptide comprising the PlySs2 lysin polypeptide orvariants thereof effective to kill gram-positive bacteria, wherein theamount of PlySs2 required to be effective to kill gram-positivebacteria, including S. aureus, in the presence of antibiotic issignificantly less than in the absence of antibiotic. The isolatedPlySs2 lysin polypeptide may comprise the amino acid sequence providedin FIG. 29 (SEQ ID NO: 1) or variants thereof having at least 80%identity, 85% identity, 90% identity, 95% identity or 99% identity tothe polypeptide of FIG. 29 (SEQ ID NO: 1) and effective to kill thegram-positive bacteria.

In an aspect of the invention, a method is provided of killinggram-positive bacteria comprising the step of contacting the bacteriawith a combination of PlySs2 lysin and one or more antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, theisolated lysin polypeptide comprising the PlySs2 lysin polypeptide orvariants thereof effective to kill gram-positive bacteria, wherein theamount of antibiotic required to be effective to kill gram-positivebacteria, including S. aureus, in the presence of PlySs2 issignificantly less than in the absence of PlySs2.

As demonstrated in accordance with the present invention, lysin asprovided herein, particularly including lysin with activity againstStaphylococcus and Streptococcus bacteria, particularly includingPlySs2, acts synergistically with antibiotics, particularly antibioticsof different class and anti-bacterial mechanism. Thus, in accordancewith the invention PlySs2 lysins or active variants thereof demonstrateenhanced activity in combination with antibiotics, including each ofantibiotics affecting cell wall synthesis such as glycopeptides,penicillins which inhibit formation of peptidoglycan, protein synthesisinhibitors, and lipopeptide antibiotic. In each instance theantibacterial activity of both lysin and antibiotic is significantlyenhanced in combination. Combination with glycopeptides antibiotic isevidenced by vancomycin, combination with penicillin class is evidencedby oxacillin, combination with protein synthesis inhibitor antibioticincluding the class of oxazolidinone is evidenced by linezolid, andcombination with lipopeptide antibiotic is evidenced by daptomycin. Thepresent invention includes and contemplates combinations and enhancedactivity with the demonstrated antibiotics as well as alternativemembers of their class or a related antibiotic.

Thus, in an aspect of the invention, a method is provided of killinggram-positive bacteria comprising the step of contacting the bacteriawith a combination of lysin and daptomycin or a related antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, whereinthe amount of daptomycin or related antibiotic required to be effectiveto kill gram-positive bacteria, including S. aureus, in the presence oflysin is significantly less than in the absence of lysin.

In a further aspect, a method is provided of killing gram-positivebacteria comprising the step of contacting the bacteria with acombination of lysin and vancomycin or a related antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, whereinthe amount of vancomycin or related antibiotic required to be effectiveto kill gram-positive bacteria, including S. aureus, in the presence oflysin is significantly less than in the absence of lysin.

In a further aspect, a method is provided of killing gram-positivebacteria comprising the step of contacting the bacteria with acombination of lysin and oxacillin or a related antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, whereinthe amount of oxacillin or related antibiotic required to be effectiveto kill gram-positive bacteria, including S. aureus, in the presence oflysin is significantly less than in the absence of lysin.

In a further aspect, a method is provided of killing gram-positivebacteria comprising the step of contacting the bacteria with acombination of lysin and linezolid or a related antibiotic, thecombination comprising an amount of an isolated lysin polypeptideeffective to kill gram-positive bacteria, including S. aureus, whereinthe amount of linezolid or related antibiotic required to be effectiveto kill gram-positive bacteria, including S. aureus, in the presence oflysin is significantly less than in the absence of lysin.

The invention also provides a method of killing antibiotic-resistantgram positive bacteria comprising contacting the antibiotic-resistantbacteria with a lysin capable of killing Staphylococcal bacteria. In onesuch aspect, the antibiotic-resistant bacteria is contacted with lysin,particularly PlySs2, in combination with an antibiotic to which thebacteria are sensitive to, or in combination with antibiotic to whichthe bacteria are resistant. In one such aspect of the method, the lysinis PLySs2. In one such aspect, the lysin is a polypeptide comprising theamino acid sequence provided in FIG. 29 (SEQ ID NO: 1) or variantsthereof having at least 80% identity, 85% identity, 90% identity, 95%identity or 99% identity to the polypeptide of FIG. 29 (SEQ ID NO: 1)and effective to kill gram-positive bacteria, particularly S. aureus.

The invention provides such a method of killing daptomycin resistantgram positive bacteria comprising contacting the daptomycin resistantbacteria with a lysin capable of killing Staphylococcal bacteria. Suchmethod may include combination of the lysin with daptomycin and/or withanother antibiotic. In one such aspect of the method, the lysin isPLySs2 as provided herein.

The invention further provides such a method of killing vancomycinresistant gram positive bacteria comprising contacting the vancomycinresistant bacteria with a lysin capable of killing Staphylococcalbacteria. Such method may include combination of the lysin withvancomycin and/or with another antibiotic. In one such aspect of themethod, the lysin is PLySs2.

In an aspect of the above methods, the methods are performed in vitro,ex vivo, or along with implantation or placement of a device in vivo soas to sterilize or decontaminate a solution, material or device,particularly intended for use by or in a human.

In a further aspect, a method is provided of enhancing antibioticeffectiveness in killing or decolonizing gram-positive bacteriacomprising the step of contacting the bacteria with a combination oflysin, particularly PlySs2, and one or more antibiotic, wherein theamount of antibiotic required to be effective to kill or decolonize thegram-positive bacteria, including S. aureus, in the presence of lysin issignificantly less than in the absence of lysin. In one such aspect, amethod is providing for enhancing or facilitating the effectiveness ofdaptomycin or a related antibiotic against Streptococcal pneumoniacomprising administering a lysin, particularly PlySs2, in combinationwith daptomycin. In a particular such method or aspect, daptomycin iseffective against Streptococcal pneumonia when administered incombination with or subsequent to administration of lysin, particularlyPlySs2, at a daptomycin dose which is ineffective in the absence oflysin, particularly PlySs2.

The invention provides a method for reducing a population ofgram-positive bacteria comprising the step of contacting the bacteriawith a composition comprising an amount of an isolated lysin polypeptideindependently ineffective to kill the gram-positive bacteria and anamount of antibiotic independently ineffective to kill the gram-positivebacteria. The antibiotic may be a glycopeptide, penicillin, proteinsynthesis inhibitor, ozalidinone or lipopeptide. Such method may includean antibiotic selected from vancomycin, daptomycin, linezolid andoxacillin. In an aspect, the isolated lysin polypeptide comprises theamino acid sequence of FIG. 29 or SEQ ID NO: 1) or variants thereofhaving at least 80% identity to the polypeptide of FIG. 29 or SEQ ID NO:1 and effective to kill the gram-positive bacteria.

The invention provides a method for reducing a population ofgram-positive bacteria comprising the step of contacting the bacteriawith a composition comprising an amount of an isolated lysin polypeptideindependently ineffective to kill the gram-positive bacteria and anamount of daptomycin independently ineffective to kill the gram-positivebacteria. In an aspect, the isolated lysin polypeptide comprises theamino acid sequence of FIG. 29 (SEQ ID NO: 1) or variants thereof havingat least 80% identity to the polypeptide of FIG. 29 (SEQ ID NO:1) andeffective to kill the gram-positive bacteria.

In any such above method or methods, the susceptible, killed, dispersedor treated bacteria may be selected from Staphylococcus aureus, Listeriamonocytogenes, Staphylococcus simulans, Streptococcus suis,Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo,Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS),Streptococcus sanguinis, Streptococcus gordonii, Streptococcusdysgalactiae, Group G Streptococcus, Group E Streptococcus, Enterococcusfaecalis and Streptococcus pneumonia.

In accordance with any of the methods of the invention, the susceptiblebacteria may be an antibiotic resistant bacteria. The bacteria may bemethicillin-resistant Staphylococcus aureus (MRSA), vancomycinintermediate-sensitivity Staphylococcus aureus (VISA), vancomycinresistant Staphylococcus aureus (VRSA), daptomycin-resistantStaphylococcus aureus (DRSA), or linezolid-resistant Staphylococcusaureus (LRSA). The susceptible bacteria may be a clinically relevant orpathogenic bacteria, particularly for humans. In an aspect of themethod(s), the lysin polyp eptide(s) is effective to killStaphylococcus, Streptococcus, Enterococcus and Listeria bacterialstrains.

In an additional aspect or embodiment of the methods and compositionsprovided herein, another distinct staphylococcal specific lysin is usedherein alone or in combination with the PlySs2 lysin as provided anddescribed herein. In one such aspect or embodiment of the methods andcompositions provided herein, the staphylococcal specific lysin ClyS isused herein alone or in combination with the PlySs2 lysin as providedand described herein.

The invention provides methods for enhancing or facilitating antibioticactivity comprising administering a combination together or in series oflysin, particularly PlySs2 lysin, and one or more antibiotic. In anaspect thereof, antibiotic activity is enhanced or facilitated by atleast 10 fold, at least 16 fold, at least 20 fold, at least 24 fold, atleast 30 fold, at least 40 fold, at least 50 fold, at least 70 fold, atleast 80 fold at least 100 fold, more than 10 fold, more than 20 fold,more than 50 fold, more than 100 fold. The invention provides methodsfor enhancing or facilitating lysin activity, particularly PlySs2 lysin,comprising administering a combination together or in series of lysin,particularly PlySs2 lysin, and one or more antibiotic. In an aspectthereof, the activity of lysin, particularly PlySs2 is enhanced at least2 fold, at least 4 fold, at least 8 fold, at least 10 fold, up to 10fold, up to 16 fold, up to 20 fold.

The invention includes a method of potentiating antibiotic activityagainst gram-positive bacteria in biological fluids havingsurfactant-like activity comprising administering antibiotic incombination with PlySs2 lysin comprising the amino acid sequenceprovided in FIG. 29 (SEQ ID NO: 1) or variants thereof having at least80% identity, 85% identity, 90% identity, 95% identity or 99% identityto the polypeptide of FIG. 29 (SEQ ID NO: 1) and effective to killgram-positive bacteria, wherein the antibiotic is effective incombination with PlySs2 at doses that the antibiotic is ineffective inthe absence of PlySs2. In an aspect of the method, the antibiotic isdaptomycin or a related compound. In an aspect, the bacteria is S.pneumoniae.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts time kill curves of various MRSA strains in the presenceof added daptomycin, vancomycin or PlySs2 lysin.

FIG. 2 depicts time kill curves of various MSSA strains in the presenceof added daptomycin, vancomycin, oxacillin or PlySs2 lysin.

FIG. 3 provides a summary plot of time kill curves of various MRSA andMSSA strains in the presence of added daptomycin, vancomycin or PlySs2lysin.

FIG. 4A-4F provides composite time kill curves of PlySS2 and antibioticson S. aureus cells in vitro. (A, B) Composite time-kill curves of PlySS2compared to oxacillin (OXA), vancomycin (VAN), and daptomycin (DAP)against sets of 20 MSSA and 42 MRSA strains, respectively. In eachindividual analysis, drug concentrations correspond to strain-specific1× MIC values. Mean values (± standard error of the mean) are shown foreach time-point. (C, D) Titration analysis of PlySS2 against sets of 15contemporary clinical MSSA and MRSA isolates, respectively. In eachindividual analysis, PlySS2 concentrations correspond to strain-specificMIC values. 4×, 1×, and 0.25× MIC concentrations were used. (E,F)Transmission electron micrographs (3300× magnification) of S. aureuscells (strain MW2) before and after 3 second treatment with 8 g/mLPlySS2. Scale bars correspond to 2 μM. Lysis results in the loss ofdarkly stained cytoplasmic components.

FIG. 5 shows time kill curves for MRSA strains treated with PlySs2 andvancomycin alone or in combination at the noted sub MIC doses.

FIG. 6 shows time kill curves for MRSA strains treated with PlySs2 anddaptomycin alone or in combination at the noted sub MIC doses.

FIG. 7 depicts time kill curves for MRSA strain 650 (O52C Tomasz) in thepresence of added daptomycin and PlySs2 lysin alone or in combination atthe noted MIC or dose.

FIG. 8A-8F shows that PlySs2 synergizes with antibiotics across multiplestrains in-vitro and depicts time-kill results for MSSA strains treatedwith PlySs2 and oxacillin (A,B); MRSA strains treated with Plyss2 andvancomycin (C,D), MRSA strains treated with PlySs2 and daptomycin (E,F).In panels A, C, and E time-kill data are shown for three individualstrains, MSSA strain JMI 7140, MRSA strain JMI 3340 and MRSA strain JMI3345 respectively. (A) Values are shown for growth, growth control (noPlySs2 or antibiotic), PlySs2 0.13× MIC, oxacillin (OXA) 0.5× MIC,PlySS2+Oxa combination of indicated drug concentrations. (C) Values areshown for growth, growth control (no PlySs2 or antibiotic), PlySs2 0.13×MIC, vancomycin (VAN) 0.5× MIC, PlySS2+VAN combination of indicated drugconcentrations. (E) Values are shown for growth, growth control (noPlySs2 or antibiotic), PlySs2 0.25× MIC, daptomycin (DAP) 0.5× MIC,PlySS2+DAP combination of indicated drug concentrations. In panels B, D,and F the log change in cfu/ml between the combination-treated cultureand the untreated growth control over 6 hours are shown for collectionsof strains. The horizontal dotted lines indicate the 2 log cutoffrequired for scoring time-kill synergy. Decreases in log₁₀ colony counts(or ΔLog₁₀ CFU/mL) are shown for cultures treated for 6 hours with drugcombination, compared to cultures treated with the most active singleagent. Synergy is defined by the CLSI as a ≥2-log₁₀ decrease in CFU/mLand is denoted in the figure by the dashed line. Key: ΔLog10CFU/mL=change in log₁₀ colony-forming units.

FIG. 9 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 553 in the presence ofreducing agent (BME).

FIG. 10 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 553 in the absence ofreducing agent (BME).

FIG. 11 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 223 in the presence ofBME.

FIG. 12 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 223 in the absence ofBME.

FIG. 13 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 270 in the presenceand absence of BME.

FIG. 14 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 269 in the presenceand absence of BME.

FIG. 15 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 241 in the presenceand absence of BME.

FIG. 16 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 263 in the presenceand absence of BME.

FIG. 17 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 650 in the presenceand absence of BME.

FIG. 18 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 828 in the presenceand absence of BME.

FIG. 19 depicts representative isobolograms depicting FIC values oflysin PlySs2 versus FIC values of antibiotic. PlySs2 versus antibioticsoxacillin, vancomycin and daptomycin are depicted against MSSA strainsand MRSA strains as noted. Oxacillin and PlySs2 are evaluated versusMSSA strain JMI 33611. PlySs2 and vancomycin are evaluated versus MSSAstrain JMI 9365 and MRSA strain JMI 6456. Daptomycin and PlySs2 areevaluated versus MSSA strain JMI 33611 and MRSA JMI 3345.

FIGS. 20A and 20B provides a time course of S. aureus staining byBODIPY-labeled daptomycin (A) and vancomycin (B) in the absence andpresence of sub-MIC amounts of PlySs2.

FIG. 21 depicts the fold change in MIC value against MRSA strain MW2 andMSSA strain ATCC 29213 treated with PlySs2 or daptomycin in the presenceof varying amounts of surfactant (from 1.25 to 15% surfactant).

FIG. 22 provides a panel of dose dilutions of pairings of daptomycin andPlySs2 at the noted concentrations on MRSA strain 269 in the presence of15% surfactant (Survanta).

FIG. 23 provides a compiled graph of % survival of mice (50 animals)challenged with MRSA strain 269 (MW2) in several experiments havingbacterial inoculum strengths of 1.1-3.1×10⁶ CFU and treated withdaptomycin or PlySs2 alone or in combination.

FIG. 24 depicts % survival of mice challenged with MRSA strain 220 at2.65×10⁶ CFU and treated with the indicated doses of daptomycin orPlySs2 alone or in combination.

FIG. 25 depicts % survival of mice challenged with MRSA strain 833 at1.4×10⁶ CFU and treated with the indicated doses of daptomycin or PlySs2alone or in combination.

FIG. 26 depicts % survival of mice challenged with MRSA strain 833 at2.0×10⁶ CFU and treated with the indicated doses of daptomycin or PlySs2alone or in combination.

FIG. 27A-27F depicts survival curves of combination therapy compared tomono-therapies in murine models of bacteremia. Mice were challenged witheither 7.5×10⁶ cfu/mouse i.p. (low challenge model—panel a) or 10⁹cfu/mouse i.p. (high challenge model—panels b-f) at time 0 and weretreated with either antibiotic, PlySs2, combination of PlySs2 andantibiotic, or control and the resulting survival data are shown inKaplan-Meier format. All doses were administered as a single bolus doseexcept for vancomycin (BID, panel e) and oxacillin (QID, panel f) whichwere administered as multiple doses over the first 24 hr period. Routesof administration were PlySs2 (i.p.), daptomycin and vancomycin(subcutaneous), and oxacillin (intramuscular). P values were calculatedfor the combinations versus antibiotic alone. (A) Low challenge modelusing MRSA strain MW2 with daptomycin at 2 mg/kg and PlySs2 at 1.25mg/kg. Dosing at 4 hr post inoculation, n=30, P<0.0001. (B) Highchallenge model using MRSA strain MW2 with daptomycin at 50 mg/kg andPlySs2 at 5.25 mg/kg. Dosing at 2 hr post inoculation, n=45, P<0.0001.(C) same as B using MRSA strain 738, n=30, P<0.0001. (D) same as B usingMRSA strain 832, n=30, P<0.0001. (E) High challenge model using MRSAstrain MW2 with vancomycin at 110 mg/kg BID and PlySs2 at 5.25 mg/kg.Dosing initiated at 2 hr post-inoculation, n=30, P<0.0001. (F) Highchallenge model using MSSA strain ATCC 25923 with oxacillin at 200 mg/kgQID and PlySs2 at 5.25 mg/kg. Dosing initiated at 2 hr post-inoculation,n=30 P<0.0001.

FIG. 28 depicts MIC of daptomycin and PlySs2 on an MSSA and a MRSAstrain with passage number and development of daptomycin resistance.PlySs2 MIC drops showing PlySs2 increased sensitivity with increaseddaptomycin resistance.

FIG. 29 provides the amino acid sequence (SEQ ID NO: 1) and encodingnucleic acid sequence (SEQ ID NO: 2) of the lysin PlySs2. The N-terminalCHAP domain and the C-terminal SH-3 domain of the PlySs2 lysin areshaded, with the CHAP domain (SEQ ID NO: 3) starting with LNN . . . andending with . . . YIT and the SH-3 domain (SEQ ID NO: 4) starting withRSY . . . and ending with . . . VAT. The CHAP domain active-siteresidues (Cys₂₆, His₁₀₂, Glu₁₁₈, and Asn₁₂₀) identified by homology toPDB 2K3A (Rossi P et al (2009) Proteins 74:515-519) are underlined.

FIG. 30 depicts fold change in daptomycin MIC value as a function ofdays of serial passage under resistance selection conditions in thepresence of daptomycin alone or daptomycin with sub-MIC amounts ofPlySs2 lysin for multiple cultures (three independent cultures of each).

FIG. 31 depicts fold change in vancomycin MIC value as a function ofdays of serial passage under resistance selection conditions in thepresence of daptomycin alone or daptomycin with sub-MIC amounts PlySs2lysin for multiple cultures (three independent cultures of each).

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]1; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “PlySs lysin(s)”, “PlySs2 lysin”, “PlySs2” and any variantsnot specifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andpresented in FIG. 29 and SEQ ID NO: 1, and the profile of activities setforth herein and in the Claims. Accordingly, proteins displayingsubstantially equivalent or altered activity are likewise contemplated.These modifications may be deliberate, for example, such asmodifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Also, the terms “PlySslysin(s)”, “PlySs2 lysin”, “PlySs2” are intended to include within theirscope proteins specifically recited herein as well as all substantiallyhomologous analogs, fragments or truncations, and allelic variations.PlySs2 lysin is described in U.S. Patent Application 61/477,836 and PCTApplication PCT/US2012/34456. A more recent paper Gilmer et al describesPlySs2 lysin (Gilmer D B et al (2013) Antimicrob Agents Chemother Epub2013 Apr. 9 [PMID 23571534]).

The term “ClyS”, “ClyS lysin” refers to a chimeric lysin ClyS, withactivity against Staphylococci bacteria, including Staphylococcusaureus, is detailed in WO 2010/002959 and also described in Daniel et al(Daniel, A et al (2010) Antimicrobial Agents and Chemother54(4):1603-1612). Exemplary ClyS amino acid sequence is provided in SEQID NO: 5.

A “lytic enzyme” includes any bacterial cell wall lytic enzyme thatkills one or more bacteria under suitable conditions and during arelevant time period. Examples of lytic enzymes include, withoutlimitation, various amidase cell wall lytic enzymes. In a particularaspect, a lytic enzyme refers to a bacteriophage lytic enzyme. A“bacteriophage lytic enzyme” refers to a lytic enzyme extracted orisolated from a bacteriophage or a synthesized lytic enzyme with asimilar protein structure that maintains a lytic enzyme functionality.

A lytic enzyme is capable of specifically cleaving bonds that arepresent in the peptidoglycan of bacterial cells to disrupt the bacterialcell wall. It is also currently postulated that the bacterial cell wallpeptidoglycan is highly conserved among most bacteria, and cleavage ofonly a few bonds to may disrupt the bacterial cell wall. Examples oflytic enzymes that cleave these bonds are muramidases, glucosaminidases,endopeptidases, or N-acetyl-muramoyl-L-alanine amidases. Fischetti et al(1974) reported that the C1 streptococcal phage lysin enzyme was anamidase. Garcia et al (1987, 1990) reported that the Cpl lysin from a S.pneumoniae from a Cp-1 phage was a lysozyme. Caldentey and Bamford(1992) reported that a lytic enzyme from the phi 6 Pseudomonas phage wasan endopeptidase, splitting the peptide bridge formed bymelo-diaminopimilic acid and D-alanine. The E. coli T1 and T6 phagelytic enzymes are amidases as is the lytic enzyme from Listeria phage(ply) (Loessner et al, 1996). There are also other lytic enzymes knownin the art that are capable of cleaving a bacterial cell wall.

A “lytic enzyme genetically coded for by a bacteriophage” includes apolypeptide capable of killing a host bacteria, for instance by havingat least some cell wall lytic activity against the host bacteria. Thepolypeptide may have a sequence that encompasses native sequence lyticenzyme and variants thereof. The polypeptide may be isolated from avariety of sources, such as from a bacteriophage (“phage”), or preparedby recombinant or synthetic methods. The polypeptide may, for example,comprise a choline-binding portion at the carboxyl terminal side and maybe characterized by an enzyme activity capable of cleaving cell wallpeptidoglycan (such as amidase activity to act on amide bonds in thepeptidoglycan) at the amino terminal side. Lytic enzymes have beendescribed which include multiple enzyme activities, for example twoenzymatic domains, such as PlyGBS lysin. Further, other lytic enzymeshave been described containing only a catalytic domain and no cell wallbinding domain.

“A native sequence phage associated lytic enzyme” includes a polypeptidehaving the same amino acid sequence as an enzyme derived from abacterial genome (i.e., a prophage). Such native sequence enzyme can beisolated or can be produced by recombinant or synthetic means.

The term “native sequence enzyme” encompasses naturally occurring forms(e.g., alternatively spliced or altered forms) and naturally-occurringvariants of the enzyme. In one embodiment of the invention, the nativesequence enzyme is a mature or full-length polypeptide that isgenetically coded for by a gene from a bacteriophage specific forStreptococcus suis. Of course, a number of variants are possible andknown, as acknowledged in publications such as Lopez et al., MicrobialDrug Resistance 3: 199-211 (1997); Garcia et al., Gene 86: 81-88 (1990);Garcia et al., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia etal., Proc. Natl. Acad. Sci. USA 85: 914-918 (1988); Garcia et al.,Streptococcal Genetics (J. J. Ferretti and Curtis eds., 1987); Lopez etal., FEMS Microbiol. Lett. 100: 439-448 (1992); Romero et al., J.Bacteriol. 172: 5064-5070 (1990); Ronda et al., Eur. J. Biochem. 164:621-624 (1987) and Sanchez et al., Gene 61: 13-19 (1987). The contentsof each of these references, particularly the sequence listings andassociated text that compares the sequences, including statements aboutsequence homologies, are specifically incorporated by reference in theirentireties.

“A variant sequence lytic enzyme” includes a lytic enzyme characterizedby a polypeptide sequence that is different from that of a lytic enzyme,but retains functional activity. The lytic enzyme can, in someembodiments, be genetically coded for by a bacteriophage specific forStreptococcus suis as in the case of PlySs2 having a particular aminoacid sequence identity with the lytic enzyme sequence(s) hereof, asprovided in FIG. 29 and in SEQ ID NO: 1. For example, in someembodiments, a functionally active lytic enzyme can kill Streptococcussuis bacteria, and other susceptible bacteria as provided herein,including as shown in TABLE 1, 2 and 3, by disrupting the cellular wallof the bacteria. An active lytic enzyme may have a 60, 65, 70, 75, 80,85, 90, 95, 97, 98, 99 or 99.5% amino acid sequence identity with thelytic enzyme sequence(s) hereof, as provided in FIG. 29 (SEQ ID NO: 1,SEQ ID NO: 3 or SEQ ID NO: 4). Such phage associated lytic enzymevariants include, for instance, lytic enzyme polypeptides wherein one ormore amino acid residues are added, or deleted at the N or C terminus ofthe sequence of the lytic enzyme sequence(s) hereof, as provided in FIG.29 (SEQ ID NO: 1).

In a particular aspect, a phage associated lytic enzyme will have atleast about 80% or 85% amino acid sequence identity with native phageassociated lytic enzyme sequences, particularly at least about 90% (e.g.90%) amino acid sequence identity. Most particularly a phage associatedlytic enzyme variant will have at least about 95% (e.g. 95%) amino acidsequence identity with the native phage associated the lytic enzymesequence(s) hereof, as provided in FIG. 29 (SEQ ID NO: 1) for PlySs2lysin, or as previously described for ClyS including in WO 2010/002959and also described in Daniel et al (Daniel, A et al (2010) AntimicrobialAgents and Chemother 54(4):1603-1612) and SEQ ID NO: 5.

“Percent amino acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified is defined herein as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the phage associated lyticenzyme sequence, after aligning the sequences in the same reading frameand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity.

“Percent nucleic acid sequence identity” with respect to the phageassociated lytic enzyme sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the phage associated lytic enzyme sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

To determine the percent identity of two nucleotide or amino acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps may be introduced in the sequence of a first nucleotidesequence). The nucleotides or amino acids at corresponding nucleotide oramino acid positions are then compared. When a position in the firstsequence is occupied by the same nucleotide or amino acid as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100).

The determination of percent identity between two sequences may beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877(1993), which is incorporated into the NBLAST program which may be usedto identify sequences having the desired identity to nucleotidesequences of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST may be utilized as described in Altschul et al.,Nucleic Acids Res, 25:3389-3402 (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,NBLAST) may be used. See the programs provided by National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health.

“Polypeptide” includes a polymer molecule comprised of multiple aminoacids joined in a linear manner. A polypeptide can, in some embodiments,correspond to molecules encoded by a polynucleotide sequence which isnaturally occurring. The polypeptide may include conservativesubstitutions where the naturally occurring amino acid is replaced byone having similar properties, where such conservative substitutions donot alter the function of the polyp eptide.

The term “altered lytic enzymes” includes shuffled and/or chimeric lyticenzymes.

Phage lytic enzymes specific for bacteria infected with a specific phagehave been found to effectively and efficiently break down the cell wallof the bacterium in question. The lytic enzyme is believed to lackproteolytic enzymatic activity and is therefore non-destructive tomammalian proteins and tissues when present during the digestion of thebacterial cell wall. Furthermore, because it has been found that theaction of phage lytic enzymes, unlike antibiotics, was rather specificfor the target pathogen(s), it is likely that the normal flora willremain essentially intact (M. J. Loessner, G. Wendlinger, S. Scherer,Mol Microbiol 16, 1231-41. (1995) incorporated herein by reference). Infact, the PlySs2 lysin, while demonstrating uniquely broad bacterialspecies and strain killing, is comparatively and particularly inactiveagainst bacteria comprising the normal flora, including E. coli, asdescribed herein.

A lytic enzyme or polypeptide of use in the invention may be produced bythe bacterial organism after being infected with a particularbacteriophage or may be produced or prepared recombinantly orsynthetically as either a prophylactic treatment for preventing thosewho have been exposed to others who have the symptoms of an infectionfrom getting sick, or as a therapeutic treatment for those who havealready become ill from the infection. In as much the lysin polypeptidesequences and nucleic acids encoding the lysin polypeptides aredescribed and referenced to herein, the lytic enzyme(s)/polypeptide(s)may be preferably produced via the isolated gene for the lytic enzymefrom the phage genome, putting the gene into a transfer vector, andcloning said transfer vector into an expression system, using standardmethods of the art, including as exemplified herein. The lytic enzyme(s)or polypeptide(s) may be truncated, chimeric, shuffled or “natural,” andmay be in combination. Relevant U.S. Pat. No. 5,604,109 is incorporatedherein in its entirety by reference. An “altered” lytic enzyme can beproduced in a number of ways. In a preferred embodiment, a gene for thealtered lytic enzyme from the phage genome is put into a transfer ormovable vector, preferably a plasmid, and the plasmid is cloned into anexpression vector or expression system. The expression vector forproducing a lysin polypeptide or enzyme of the invention may be suitablefor E. coli, Bacillus, or a number of other suitable bacteria. Thevector system may also be a cell free expression system. All of thesemethods of expressing a gene or set of genes are known in the art. Thelytic enzyme may also be created by infecting Streptococcus suis with abacteriophage specific for Streptococcus suis, wherein said at least onelytic enzyme exclusively lyses the cell wall of said Streptococcus suishaving at most minimal effects on other, for example natural orcommensal, bacterial flora present.

A “chimeric protein” or “fusion protein” comprises all or (preferably abiologically active) part of a polypeptide of use in the inventionoperably linked to a heterologous polypeptide. Chimeric proteins orpeptides are produced, for example, by combining two or more proteinshaving two or more active sites. Chimeric protein and peptides can actindependently on the same or different molecules, and hence have apotential to treat two or more different bacterial infections at thesame time. Chimeric proteins and peptides also may be used to treat abacterial infection by cleaving the cell wall in more than one location,thus potentially providing more rapid or effective (or synergistic)killing from a single lysin molecule or chimeric peptide.

A “heterologous” region of a DNA construct or peptide construct is anidentifiable segment of DNA within a larger DNA molecule or peptidewithin a larger peptide molecule that is not found in association withthe larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA or peptide as defined herein.

The term “operably linked” means that the polypeptide of the disclosureand the heterologous polypeptide are fused in-frame. The heterologouspolypeptide can be fused to the N-terminus or C-terminus of thepolypeptide of the disclosure. Chimeric proteins are producedenzymatically by chemical synthesis, or by recombinant DNA technology. Anumber of chimeric lytic enzymes have been produced and studied. Oneexample of a useful fusion protein is a GST fusion protein in which thepolypeptide of the disclosure is fused to the C-terminus of a GSTsequence. Such a chimeric protein can facilitate the purification of arecombinant polypeptide of the disclosure.

In another embodiment, the chimeric protein or peptide contains aheterologous signal sequence at its N-terminus. For example, the nativesignal sequence of a polypeptide of the disclosure can be removed andreplaced with a signal sequence from another known protein.

The fusion protein may combine a lysin polypeptide with a protein orpolypeptide of having a different capability, or providing an additionalcapability or added character to the lysin polypeptide. The fusionprotein may be an immunoglobulin fusion protein in which all or part ofa polypeptide of the disclosure is fused to sequences derived from amember of the immunoglobulin protein family. The immunoglobulin may bean antibody, for example an antibody directed to a surface protein orepitope of a susceptible or target bacteria. The immunoglobulin fusionprotein can alter bioavailability of a cognate ligand of a polypeptideof the disclosure. Inhibition of ligand/receptor interaction may beuseful therapeutically, both for treating bacterial-associated diseasesand disorders for modulating (i.e. promoting or inhibiting) cellsurvival. The fusion protein may include a means to direct or target thelysin, including to particular tissues or organs or to surfaces such asdevices, plastic, membranes. Chimeric and fusion proteins and peptidesof the disclosure can be produced by standard recombinant DNAtechniques.

A modified or altered form of the protein or peptides and peptidefragments, as disclosed herein, includes protein or peptides and peptidefragments that are chemically synthesized or prepared by recombinant DNAtechniques, or both. These techniques include, for example,chimerization and shuffling. As used herein, shuffled proteins orpeptides, gene products, or peptides for more than one related phageprotein or protein peptide fragments have been randomly cleaved andreassembled into a more active or specific protein. Shuffledoligonucleotides, peptides or peptide fragment molecules are selected orscreened to identify a molecule having a desired functional property.Shuffling can be used to create a protein that is more active, forinstance up to 10 to 100 fold more active than the template protein. Thetemplate protein is selected among different varieties of lysinproteins. The shuffled protein or peptides constitute, for example, oneor more binding domains and one or more catalytic domains. When theprotein or peptide is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. Accordingly such preparationsof the protein have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.

The present invention also pertains to other variants of thepolypeptides useful in the invention. Such variants may have an alteredamino acid sequence which can function as either agonists (mimetics) oras antagonists. Variants can be generated by mutagenesis, i.e., discretepoint mutation or truncation. An agonist can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the protein. An antagonist of a protein can inhibitone or more of the activities of the naturally occurring form of theprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein. Variants of a protein of use in the disclosure which functionas either agonists (mimetics) or as antagonists can be identified byscreening combinatorial libraries of mutants, such as truncationmutants, of the protein of the disclosure. In one embodiment, avariegated library of variants is generated by combinatorial mutagenesisat the nucleic acid level and is encoded by a variegated gene library.There are a variety of methods which can be used to produce libraries ofpotential variants of the polypeptides of the disclosure from adegenerate oligonucleotide sequence. Libraries of fragments of thecoding sequence of a polypeptide of the disclosure can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants, active fragments or truncations.Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. In this context, the smallest portion of a protein(or nucleic acid that encodes the protein) according to embodiments isan epitope that is recognizable as specific for the phage that makes thelysin protein. Accordingly, the smallest polypeptide (and associatednucleic acid that encodes the polypeptide) that can be expected to binda target or receptor, such as an antibody, and is useful for someembodiments may be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 75, 85, or 100 amino acids long. Although small sequences asshort as 8, 9, 10, 11, 12 or 15 amino acids long reliably compriseenough structure to act as targets or epitopes, shorter sequences of 5,6, or 7 amino acids long can exhibit target or epitopic structure insome conditions and have value in an embodiment. Thus, the smallestportion of the protein(s) or lysin polypeptides provided herein,including as set out in FIG. 29 (SEQ ID NO: 1) includes polypeptides assmall as 5, 6, 7, 8, 9, 10, 12, 14 or 16 amino acids long.

Biologically active portions of a protein or peptide fragment of theembodiments, as described herein, include polypeptides comprising aminoacid sequences sufficiently identical to or derived from the amino acidsequence of the lysin protein of the disclosure, which include feweramino acids than the full length protein of the lysin protein andexhibit at least one activity of the corresponding full-length protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the corresponding protein. An exemplary domainsequence for the N terminal CHAP domain of the lysin of the presentinvention is provided in FIG. 29 and SEQ ID NO: 3. An exemplary domainsequence for the C terminal SH3 domain of the lysin of the presentinvention is provided in FIG. 29 and SEQ ID NO: 4. A biologically activeportion of a protein or protein fragment of the disclosure can be apolypeptide which is, for example, 10, 25, 50, 100 less or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, or added can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of the native form of a polypeptide of the embodiments.

Homologous proteins and nucleic acids can be prepared that sharefunctionality with such small proteins and/or nucleic acids (or proteinand/or nucleic acid regions of larger molecules) as will be appreciatedby a skilled artisan. Such small molecules and short regions of largermolecules that may be homologous specifically are intended asembodiments. Preferably the homology of such valuable regions is atleast 50%, 65%, 75%, 80%, 85%, and preferably at least 90%, 95%, 97%,98%, or at least 99% compared to the lysin polypeptides provided herein,including as set out in FIG. 29. These percent homology values do notinclude alterations due to conservative amino acid substitutions.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, atleast about 85%, and preferably at least about 90 or 95%) are identical,or represent conservative substitutions. The sequences of comparablelysins, such as comparable PlySs2 lysins, or comparable ClyS lysins, aresubstantially homologous when one or more, or several, or up to 10%, orup to 15%, or up to 20% of the amino acids of the lysin polypeptide aresubstituted with a similar or conservative amino acid substitution, andwherein the comparable lysins have the profile of activities,anti-bacterial effects, and/or bacterial specificities of a lysin, suchas the PlySs2 lysin and/or ClyS lysin, disclosed herein.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

Mutations can be made in the amino acid sequences, or in the nucleicacid sequences encoding the polypeptides and lysins herein, including inthe lysin sequences set out in FIG. 29 (SEQ ID NO: 1), or in activefragments or truncations thereof, such that a particular codon ischanged to a codon which codes for a different amino acid, an amino acidis substituted for another amino acid, or one or more amino acids aredeleted. Such a mutation is generally made by making the fewest aminoacid or nucleotide changes possible. A substitution mutation of thissort can be made to change an amino acid in the resulting protein in anon-conservative manner (for example, by changing the codon from anamino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to another grouping)or in a conservative manner (for example, by changing the codon from anamino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to the same grouping).Such a conservative change generally leads to less change in thestructure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein.

Thus, one of skill in the art, based on a review of the sequence of thePlySs2 lysin polypeptide provided herein and on their knowledge and thepublic information available for other lysin polypeptides, can makeamino acid changes or substitutions in the lysin polypeptide sequence.Amino acid changes can be made to replace or substitute one or more, oneor a few, one or several, one to five, one to ten, or such other numberof amino acids in the sequence of the lysin(s) provided herein togenerate mutants or variants thereof. Such mutants or variants thereofmay be predicted for function or tested for function or capability forkilling bacteria, including Staphylococcal, Streptococcal, Listeria, orEnterococcal bacteria, and/or for having comparable activity to thelysin(s) as described and particularly provided herein. Thus, changescan be made to the sequence of lysin, and mutants or variants having achange in sequence can be tested using the assays and methods describedand exemplified herein, including in the examples. One of skill in theart, on the basis of the domain structure of the lysin(s) hereof canpredict one or more, one or several amino acids suitable forsubstitution or replacement and/or one or more amino acids which are notsuitable for substitution or replacement, including reasonableconservative or non-conservative substitutions.

In this regard, and with exemplary reference to PlySs2 lysin it ispointed out that, although the PlySs2 polypeptide lysin represents adivergent class of prophage lytic enzyme, the lysin comprises anN-terminal CHAP domain (cysteine-histidine amidohydrolase/peptidase)(SEQ ID NO: 3) and a C-terminal SH3-type 5 domain (SEQ ID NO: 4) asdepicted in FIG. 29. The domains are depicted in the amino acid sequencein distinct shaded color regions, with the CHAP domain corresponding tothe first shaded amino acid sequence region starting with LNN . . . andthe SH3-type 5 domain corresponding to the second shaded region startingwith RSY . . . CHAP domains are included in several previouslycharacterized streptococcal and staphylococcal phage lysins. Thus, oneof skill in the art can reasonably make and test substitutions orreplacements to the CHAP domain and/or the SH-3 domain of PlySs2.Sequence comparisons to the Genbank database can be made with either orboth of the CHAP and/or SH-3 domain sequences or with the PlySs2 lysinfull amino acid sequence, for instance, to identify amino acids forsubstitution. For example, the CHAP domain contains conserved cysteineand histidine amino acid sequences (the first cysteine and histidine inthe CHAP domain) which are characteristic and conserved in CHAP domainsof different polypeptides. It is reasonable to predict, for example,that the conserved cysteine and histidine residues should be maintainedin a mutant or variant of PlySs2 so as to maintain activity orcapability. It is notable that a mutant or variant having an alaninereplaced for valine at valine amino acid 19 in the PlySs2 amino acidsequence of FIG. 29 (SEQ ID NO: 1) is active and capable of killing grampositive bacteria in a manner similar to and as effective as the FIG. 29(SEQ ID NO: 1) PlySs2 lysin.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)

Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Exemplary and preferred conservative amino acid substitutions includeany of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L)for valine (V) and vice versa; serine (S) for threonine (T) and viceversa; isoleucine (I) for valine (V) and vice versa; lysine (K) forglutamine (Q) and vice versa; isoleucine (I) for methionine (M) and viceversa; serine (S) for asparagine (N) and vice versa; leucine (L) formethionine (M) and vice versa; lysine (L) for glutamic acid (E) and viceversa; alanine (A) for serine (S) and vice versa; tyrosine (Y) forphenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid(D) and vice versa; leucine (L) for isoleucine (I) and vice versa;lysine (K) for arginine (R) and vice versa.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

In accordance with the present invention compositions and methods areprovide based on combinations of bacteriophage lysin(s) with antibioticare provided for rapid and effective killing of gram positive bacteria.In accordance with the invention, the lysin PlySs2, which demonstratesbroad killing activity against multiple bacteria, particularlygram-positive bacteria, including Staphylococcus and Streptococcusbacterial strains, provides remarkable synergy in combination withantibiotic(s) and can significantly reduce the effective MIC dosesrequired for antibiotic(s).

As demonstrated and provided herein, lysin particularly PlySs2 lysin iscapable of synergizing with antibiotics, including antibiotics ofdifferent types and classes, including vancomycin, daptomycin,linezolid, and oxacillin, in a process characterized by improvedbactericidal activity, more rapid antibiotic penetration, andsuppression of resistance. In murine bacteremia models, as demonstratedherein, pair-wise combinations of PlySs2 with antibiotics confer ahighly significant survival increase relative to single-agenttreatments. Thus, lysin/antibiotic combinations, relative to currentstandard treatments, will be more effective therapies for treatingbacteremia in the clinic.

The invention further demonstrates PlySs2-dependent enhancement ofantibiotics in combination via both in-vitro assays and in a murinemodel of S. aureus-induced bacteremia under conditions in whichhuman-simulated doses of single-agent antibiotics fail. Data arepresented herein illustrating the mechanism of the PlySs2-mediatedenhancement of antibiotic activity and indicating a general synergybetween lysins and antibiotics. Synergy has implications for anefficacious new general anti-infective strategy based on theco-administration of lysin and antibiotics. In particular each and bothagents lysins and antibiotics may be administered at significantlyreduced doses and amounts, with enhanced bacteriocidal andbacteriostatic activity and with reduced risk of antibiotic or agentresistance.

While lysin, particularly PlySs2 lysin, is recognized as a single agent,the present invention provides that lysin, particularly PlySs2 lysin,remarkably demonstrates a significant degree of in vitro and in vivosynergy with various antibiotics. While in the present Examples synergyis validated by time-kill curves and checkerboard assays with multiplestrains and antibiotics, the extent of in vitro synergy is particularlyillustrated using a dual agent MIC assay in which as little as 0.25× MICPlySs2 reduced the daptomycin MIC from 1 μg/mL to 0.0075 μg/mL, a128-fold decrease. This synergistic effect was seen across 12 MRSAstrains with the degree of potency enhancement ranging from 64 to256-fold. The two antimicrobials, antibiotics plus lysin, in acombination are therefore doing more than simply killing sequentially(reduction of the bulk population by lysin followed by antibiotickilling of residual bacteria) since 7.5 ng/ml daptomycin is vastlyinsufficient to kill as a single agent.

In the bacteremia models provided and demonstrated herein, combinationtherapy treatments consistently outperformed full strengthhuman-simulated doses of single agent antibiotic treatments. This isdemonstrated for both vancomycin and daptomycin, the currentstandard-of-care antibiotics for treating MRSA bacteremia, as well asfor oxacillin, a beta-lactam, the current standard-of-care antibioticfor treating MSSA bacteremia. These results have clear clinicalimplications and provide new effective combination therapy regimensemploying lysin(s) and antibiotic(s) for treating bacteremia as well asother serious infections. Provided are methods and compositions based oncombination lysin plus antibiotic therapy using lower doses of theseagents with enhanced efficacy and lower risk of resistance. Indeed thepresent methods and compositions are effective on resistant bacteria,including antibiotic resistant Staphylococcal bacteria.

In clinical applications, the invention provides methods of treatingbacteremia by administering a lysin/antibiotic combination, particularlyPlySs2/antibiotic combination. While above its MIC, the fast-actinglysin will effectively reduce the pathogen population. Once the lysinconcentration falls below the MIC, the combination partner antibiotic'sactivity will be enhanced synergistically by the presence of the lysinfor approximately one or two more lysin pharmacokinetic half-livesextending the time in which synergy-enhanced killing is active. Thus,PlySs2/antibiotic combinations will provide more potent and effectiveantibacterial therapies than the currently available single-agentoptions.

The PlySs2 lysin displays activity and capability to kill numerousdistinct strains and species of gram positive bacteria, includingStaphylococcal, Streptococcal, Listeria, or Enterococcal bacteria. Inparticular and with significance, PlySs2 is active in killingStaphylococcus strains, including Staphylococcus aureus, particularlyboth antibiotic-sensitive and distinct antibiotic-resistant strains.PlySs2 is also active in killing Streptococcus strains, and showsparticularly effective killing against Group A and Group B streptococcusstrains. PlySs2 lysin capability against bacteria is depicted below inTABLE 1, based on log kill assessments using isolated strains in vitro.

TABLE 1 PlySs2 Reduction in Growth of Different Bacteria (partiallisting) Bacteria Relative Kill with PlySs2 Staphylococcus aureus +++(VRSA, VISA, MRSA, MSSA) Streptococcus suis +++ Staphylococcusepidermidis ++ Staphylococcus simulans +++ Lysteria monocytogenes ++Enterococcus faecalis ++ Streptococcus dysgalactiae - GBS ++Streptococcus agalactiae - GBS +++ Streptococcus pyogenes - GAS +++Streptococcus equi ++ Streptococcus sanguinis ++ Streptococcus gordonii++ Streptococcus sobrinus + Streptococcus rattus + Streptococcusoralis + Streptococcus pneumonine + Bacillus thuringiensis − Bacilluscereus − Bacillus subtilis − Bacillus anthracis − Escherichia coli −Enterococcus faecium − Pseudomanas aeruginosa −

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show significant binding tomolecules other than its specific binding partner(s). The term is alsoapplicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is tosay permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention hereof, those of skill in the art will appreciate thatminor modifications to the N- or C-terminal of the peptide may howeverbe contemplated, such as the chemical modification of the terminal toadd a protecting group or the like, e.g. the amidation of theC-terminus.

The term “isolated” refers to the state in which the lysinpolypeptide(s) of the invention, or nucleic acid encoding suchpolypeptides will be, in accordance with the present invention.Polypeptides and nucleic acid will be free or substantially free ofmaterial with which they are naturally associated such as otherpolypeptides or nucleic acids with which they are found in their naturalenvironment, or the environment in which they are prepared (e.g. cellculture) when such preparation is by recombinant DNA technologypracticed in vitro or in vivo. Polypeptides and nucleic acid may beformulated with diluents or adjuvants and still for practical purposesbe isolated—for example the polypeptides will normally be mixed withpolymers or mucoadhesives or other carriers, or will be mixed withpharmaceutically acceptable carriers or diluents, when used in diagnosisor therapy.

Nucleic acids capable of encoding the S. suis PlySs2 lysinpolypeptide(s) useful and applicable in the invention are providedherein. Representative nucleic acid sequences in this context arepolynucleotide sequences coding for the polypeptide of FIG. 29 (SEQ IDNO: 1), and sequences that hybridize, under stringent conditions, withcomplementary sequences of the DNA of the FIG. 29 (SEQ ID NO: 2)sequence(s). Further variants of these sequences and sequences ofnucleic acids that hybridize with those shown in the figures also arecontemplated for use in production of lysing enzymes according to thedisclosure, including natural variants that may be obtained. A largevariety of isolated nucleic acid sequences or cDNA sequences that encodephage associated lysing enzymes and partial sequences that hybridizewith such gene sequences are useful for recombinant production of thelysin enzyme(s) or polypeptide(s) of the invention.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

DNA molecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and which differ from those disclosed bythe deletion, addition or substitution of nucleotides while stillencoding a protein which possesses the functional characteristic of thelysin polypeptide(s) are contemplated by the disclosure. Also includedare small DNA molecules which are derived from the disclosed DNAmolecules. Such small DNA molecules include oligonucleotides suitablefor use as hybridization probes or polymerase chain reaction (PCR)primers. As such, these small DNA molecules will comprise at least asegment of a lytic enzyme genetically coded for by a bacteriophage ofStaphylococcus suis and, for the purposes of PCR, will comprise at leasta 10-15 nucleotide sequence and, more preferably, a 15-30 nucleotidesequence of the gene. DNA molecules and nucleotide sequences which arederived from the disclosed DNA molecules as described above may also bedefined as DNA sequences which hybridize under stringent conditions tothe DNA sequences disclosed, or fragments thereof.

In preferred embodiments of the present disclosure, stringent conditionsmay be defined as those under which DNA molecules with more than 25%sequence variation (also termed “mismatch”) will not hybridize. In amore preferred embodiment, stringent conditions are those under whichDNA molecules with more than 15% mismatch will not hybridize, and morepreferably still, stringent conditions are those under which DNAsequences with more than 10% mismatch will not hybridize. Preferably,stringent conditions are those under which DNA sequences with more than6% mismatch will not hybridize.

The degeneracy of the genetic code further widens the scope of theembodiments as it enables major variations in the nucleotide sequence ofa DNA molecule while maintaining the amino acid sequence of the encodedprotein. Thus, the nucleotide sequence of the gene could be changed atthis position to any of these three codons without affecting the aminoacid composition of the encoded protein or the characteristics of theprotein. The genetic code and variations in nucleotide codons forparticular amino acids are well known to the skilled artisan. Based uponthe degeneracy of the genetic code, variant DNA molecules may be derivedfrom the cDNA molecules disclosed herein using standard DNA mutagenesistechniques as described above, or by synthesis of DNA sequences. DNAsequences which do not hybridize under stringent conditions to the cDNAsequences disclosed by virtue of sequence variation based on thedegeneracy of the genetic code are herein comprehended by thisdisclosure.

Thus, it should be appreciated that also within the scope of the presentinvention are DNA sequences encoding a lysin of the present invention,including PlySs2 and PlySs1, which sequences code for a polypeptidehaving the same amino acid sequence as provided in FIG. 29 (SEQ ID NO:1), but which are degenerate thereto or are degenerate to the exemplarynucleic acids sequences provided in FIG. 29 (SEQ ID NO: 2). By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art the codonswhich can be used interchangeably to code for each specific amino acid.

One skilled in the art will recognize that the DNA mutagenesistechniques described here and known in the art can produce a widevariety of DNA molecules that code for a bacteriophage lysin ofStreptococcus suis yet that maintain the essential characteristics ofthe lytic polypeptides described and provided herein. Newly derivedproteins may also be selected in order to obtain variations on thecharacteristic of the lytic polypeptide(s), as will be more fullydescribed below. Such derivatives include those with variations in aminoacid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation may bepredetermined, the mutation per se does not need to be predetermined.Amino acid substitutions are typically of single residues, or can be ofone or more, one or a few, one, two, three, four, five, six or sevenresidues; insertions usually will be on the order of about from 1 to 10amino acid residues; and deletions will range about from 1 to 30residues. Deletions or insertions may be in single form, but preferablyare made in adjacent pairs, i.e., a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. Substitutionalvariants are those in which at least one residue in the amino acidsequence has been removed and a different residue inserted in its place.Such substitutions may be made so as to generate no significant effecton the protein characteristics or when it is desired to finely modulatethe characteristics of the protein. Amino acids which may be substitutedfor an original amino acid in a protein and which are regarded asconservative substitutions are described above and will be recognized byone of skill in the art.

As is well known in the art, DNA sequences may be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host. Such operative linking of aDNA sequence of this invention to an expression control sequence, ofcourse, includes, if not already part of the DNA sequence, the provisionof an initiation codon, ATG, in the correct reading frame upstream ofthe DNA sequence. A wide variety of host/expression vector combinationsmay be employed in expressing the DNA sequences of this invention.Useful expression vectors, for example, may consist of segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Any of a widevariety of expression control sequences—sequences that control theexpression of a DNA sequence operatively linked to it—may be used inthese vectors to express the DNA sequences of this invention. A widevariety of unicellular host cells are also useful in expressing the DNAsequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, human cells and plant cells in tissue culture. One skilled in theart will be able to select the proper vectors, expression controlsequences, and hosts without undue experimentation to accomplish thedesired expression without departing from the scope of this invention.

Therapeutic or pharmaceutical compositions comprising the lyticenzyme(s)/polypeptide(s) of use in the methods and applications providedin the invention are provided herein, as well as related methods of use.Therapeutic or pharmaceutical compositions may comprise one or morelytic polypeptide(s), and optionally include natural, truncated,chimeric or shuffled lytic enzymes, combined with one or moreantibiotics, optionally combined with suitable excipients, carriers orvehicles. The invention provides therapeutic compositions orpharmaceutical compositions of the lysins, including PlySs2, incombination with antibiotic for use in the killing, alleviation,decolonization, prophylaxis or treatment of gram-positive bacteria,including bacterial infections or related conditions. The inventionprovides therapeutic compositions or pharmaceutical compositions of thelysins, including PlySs2, in combination with vancomycin, linezolid ordaptomycin for use in the killing, alleviation, decolonization,prophylaxis or treatment of gram-positive bacteria, including bacterialinfections or related conditions. The invention provides therapeuticcompositions or pharmaceutical compositions of the lysins, includingPlySs2, in combination with daptomycin for use in the killing,alleviation, decolonization, prophylaxis or treatment of gram-positivebacteria, including bacterial infections or related conditions.Compositions comprising PlySs2 lysin, including truncations or variantsthereof, in combination with antibiotic, including daptomycin, areprovided herein for use in the killing, alleviation, decolonization,prophylaxis or treatment of gram-positive bacteria, including bacterialinfections or related conditions, particularly of Streptococcus,Staphylococcus, Enterococcus or Listeria, including Streptococcuspyogenes and antibiotic resistant Staphylococcus aureus.

The enzyme(s) or polypeptide(s) included in the therapeutic compositionsmay be one or more or any combination of unaltered phage associatedlytic enzyme(s), truncated lytic polypeptides, variant lyticpolypeptide(s), and chimeric and/or shuffled lytic enzymes.Additionally, different lytic polypeptide(s) genetically coded for bydifferent phage for treatment of the same bacteria may be used. Theselytic enzymes may also be any combination of “unaltered” lytic enzymesor polypeptides, truncated lytic polypeptide(s), variant lyticpolypeptide(s), and chimeric and shuffled lytic enzymes. The lyticenzyme(s)/polypeptide(s) in a therapeutic or pharmaceutical compositionfor gram-positive bacteria, including Streptococcus, Staphylococcus,Enterococcus and Listeria, may be used alone or in combination withantibiotics or, if there are other invasive bacterial organisms to betreated, in combination with other phage associated lytic enzymesspecific for other bacteria being targeted. The lytic enzyme, truncatedenzyme, variant enzyme, chimeric enzyme, and/or shuffled lytic enzymemay be used in conjunction with a holin protein. The amount of the holinprotein may also be varied. Various antibiotics may be optionallyincluded in the therapeutic composition with the enzyme(s) orpolypeptide(s) and with or without the presence of lysostaphin. Morethan one lytic enzyme or polypeptide may be included in the therapeuticcomposition.

The pharmaceutical composition can also include one or more alteredlytic enzymes, including isozymes, analogs, or variants thereof,produced by chemical synthesis or DNA recombinant techniques. Inparticular, altered lytic protein can be produced by amino acidsubstitution, deletion, truncation, chimerization, shuffling, orcombinations thereof. The pharmaceutical composition may contain acombination of one or more natural lytic protein and one or moretruncated, variant, chimeric or shuffled lytic protein. Thepharmaceutical composition may also contain a peptide or a peptidefragment of at least one lytic protein derived from the same ordifferent bacteria species, with an optional addition of one or morecomplementary agent, and a pharmaceutically acceptable carrier ordiluent.

The pharmaceutical compositions of the present invention contain acomplementary agent—one or more conventional antibiotics—particularly asprovided herein. Antibiotics can be subgrouped broadly into thoseaffecting cell wall peptidoglycan biosynthesis and those affecting DNAor protein synthesis in gram positive bacteria. Cell wall synthesisinhibitors, including penicillin and antibiotics like it, disrupt therigid outer cell wall so that the relatively unsupported cell swells andeventually ruptures. The complementary agent may be an antibiotic, suchas erythromycin, clarithromycin, azithromycin, roxithromycin, othermembers of the macrolide family, penicilins, cephalosporins, and anycombinations thereof in amounts which are effective to synergisticallyenhance the therapeutic effect of the lytic enzyme. Virtually any otherantibiotic may be used with the altered and/or unaltered lytic enzyme.Antibiotics affecting cell wall peptidoglycan biosynthesis include:Glycopeptides, which inhibit peptidoglycan synthesis by preventing theincorporation of N-acetylmuramic acid (NAM) and N-acetylglucosamine(NAG) peptide subunits into the peptidoglycan matrix. Availableglycopeptides include vancomycin and teicoplanin; Penicillins, which actby inhibiting the formation of peptidoglycan cross-links. The functionalgroup of penicillins, the β-lactam moiety, binds and inhibitsDD-transpeptidase that links the peptidoglycan molecules in bacteria.Hydrolytic enzymes continue to break down the cell wall, causingcytolysis or death due to osmotic pressure. Common penicillins includeoxacillin, ampicillin and cloxacillin; and Polypeptides, which interferewith the dephosphorylation of the C₅₅-isoprenyl pyrophosphate, amolecule that carries peptidoglycan building-blocks outside of theplasma membrane. A cell wall-impacting polypeptide is bacitracin. Otheruseful and relevant antibiotics include vancomycin, linezolid, anddaptomycin.

Other lytic enzymes may be included in the carrier to treat otherbacterial infections. The pharmaceutical composition can also contain apeptide or a peptide fragment of at least one lytic protein, one holinprotein, or at least one holin and one lytic protein, which lytic andholin proteins are each derived from the same or different bacteriaspecies, with an optional addition of a complementary agents, and asuitable carrier or diluent.

Also provided are compositions containing nucleic acid molecules that,either alone or in combination with other nucleic acid molecules, arecapable of expressing an effective amount of a lytic polypeptide(s) or apeptide fragment of a lytic polypeptide(s) in vivo. Cell culturescontaining these nucleic acid molecules, polynucleotides, and vectorscarrying and expressing these molecules in vitro or in vivo, are alsoprovided.

Therapeutic or pharmaceutical compositions may comprise lyticpolypeptide(s) and antibiotic(s) combined with a variety of carriers totreat the illnesses caused by the susceptible gram-positive bacteria.The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts. Glycerin or glycerol(1,2,3-propanetriol) is commercially available for pharmaceutical use.DMSO is an aprotic solvent with a remarkable ability to enhancepenetration of many locally applied drugs. The carrier vehicle may alsoinclude Ringer's solution, a buffered solution, and dextrose solution,particularly when an intravenous solution is prepared.

The effective dosage rates or amounts of an altered or unaltered lyticenzyme/polypeptide(s) of and for use in the present invention willdepend in part on whether the lytic enzyme/polypeptide(s) will be usedtherapeutically or prophylactically, the duration of exposure of therecipient to the infectious bacteria, the size and weight of theindividual, etc. The duration for use of the composition containing theenzyme/polypeptide(s) also depends on whether the use is forprophylactic purposes, wherein the use may be hourly, daily or weekly,for a short time period, or whether the use will be for therapeuticpurposes wherein a more intensive regimen of the use of the compositionmay be needed, such that usage may last for hours, days or weeks, and/oron a daily basis, or at timed intervals during the day. Any dosage formemployed should provide for a minimum number of units for a minimumamount of time. Carriers that are classified as “long” or “slow” releasecarriers (such as, for example, certain nasal sprays or lozenges) couldpossess or provide a lower concentration of active (enzyme) units perml, but over a longer period of time, whereas a “short” or “fast”release carrier (such as, for example, a gargle) could possess orprovide a high concentration of active (enzyme) units per ml, but over ashorter period of time. The amount of active units per ml and theduration of time of exposure depend on the nature of infection, whethertreatment is to be prophylactic or therapeutic, and other variables.There are situations where it may be necessary to have a much higherunit/ml dosage or a lower unit/ml dosage.

The lytic enzyme/polypeptide(s) should be in an environment having a pHwhich allows for activity of the lytic enzyme/polypeptide(s). Astabilizing buffer may allow for the optimum activity of the lysinenzyme/polypeptide(s). The buffer may contain a reducing reagent, suchas dithiothreitol or beta mercaptoethanol (BME). The stabilizing buffermay also be or include a metal chelating reagent, such asethylenediaminetetracetic acid disodium salt, or it may also contain aphosphate or citrate-phosphate buffer, or any other buffer.

It is notable that the environment and certain aspects of the treatmentlocation can affect the effectiveness of an antibiotic. For instance,daptomycin binds avidly to pulmonary surfactant and therefore is noteffective in treatment of bacterial pneumonia, including staphylococcalpneumonia. The present invention demonstrates the remarkableeffectiveness and synergy of PlySs2 and daptomycin in combinationagainst susceptible bacteria. In addition, PlySs2 lysin and daptomycinin combination remain very effective in the presence of a commerciallyavailable surfactant which mimics pulmonary surfactant. Thus, PlySs2facilitates and enhances the effectiveness of antibiotic, particularlydaptomycin, and serves to enable daptomycin effectiveness and activityeven in the presence of surfactant.

A mild surfactant can be included in a therapeutic or pharmaceuticalcomposition in an amount effective to potentiate the therapeutic effectof the lytic enzyme/polypeptide(s) may be used in a composition.Suitable mild surfactants include, inter alia, esters of polyoxyethylenesorbitan and fatty acids (Tween series), octylphenoxy polyethoxy ethanol(Triton-X series), n-Octyl-.beta.-D-glucopyranoside,n-Octyl-.beta.-D-thioglucopyranoside, n-Decyl-.beta.-D-glucopyranoside,n-Dodecyl-.beta.-D-glucopyranoside, and biologically occurringsurfactants, e.g., fatty acids, glycerides, monoglycerides, deoxycholateand esters of deoxycholate.

Preservatives may also be used in this invention and preferably compriseabout 0.05% to 0.5% by weight of the total composition. The use ofpreservatives assures that if the product is microbially contaminated,the formulation will prevent or diminish microorganism growth. Somepreservatives useful in this invention include methylparaben,propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDMHydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate,chlorhexidine digluconate, or a combination thereof.

The therapeutic composition may further comprise other enzymes, such asthe enzyme lysostaphin for the treatment of any Staphylococcus aureusbacteria present along with the susceptible gram-positive bacteria.Lysostaphin, a gene product of Staphylococcus simulans, exerts abacteriostatic and bactericidal effect upon S. aureus by enzymaticallydegrading the polyglycine crosslinks of the cell wall (Browder et al.,Res. Comm., 19: 393-400 (1965)). The gene for lysostaphin hassubsequently been cloned and sequenced (Recsei et al., Proc. Natl. Acad.Sci. USA, 84: 1127-1131 (1987). A therapeutic composition may alsoinclude mutanolysin, and lysozyme.

Means of application of the therapeutic composition comprising a lyticenzyme/polypeptide(s) and antibiotic(s) include, but are not limited todirect, indirect, carrier and special means or any combination of means.Direct application of the lytic enzyme/polypeptide(s) may be by anysuitable means to directly bring the polypeptide in contact with thesite of infection or bacterial colonization, such as to the nasal area(for example nasal sprays), dermal or skin applications (for exampletopical ointments or formulations), suppositories, tampon applications,etc. Nasal applications include for instance nasal sprays, nasal drops,nasal ointments, nasal washes, nasal injections, nasal packings,bronchial sprays and inhalers, or indirectly through use of throatlozenges, mouthwashes or gargles, or through the use of ointmentsapplied to the nasal nares, or the face or any combination of these andsimilar methods of application. The forms in which the lytic enzyme maybe administered include but are not limited to lozenges, troches,candies, injectants, chewing gums, tablets, powders, sprays, liquids,ointments, and aerosols.

The mode of application for the lytic enzyme and antibiotic includes anumber of different types and combinations of carriers which include,but are not limited to an aqueous liquid, an alcohol base liquid, awater soluble gel, a lotion, an ointment, a nonaqueous liquid base, amineral oil base, a blend of mineral oil and petrolatum, lanolin,liposomes, protein carriers such as serum albumin or gelatin, powderedcellulose carmel, and combinations thereof. A mode of delivery of thecarrier containing the therapeutic agent includes, but is not limited toa smear, spray, a time-release patch, a liquid absorbed wipe, andcombinations thereof. The lytic enzyme may be applied to a bandageeither directly or in one of the other carriers. The bandages may besold damp or dry, wherein the enzyme is in a lyophilized form on thebandage. This method of application is most effective for the treatmentof infected skin. The carriers of topical compositions may comprisesemi-solid and gel-like vehicles that include a polymer thickener,water, preservatives, active surfactants or emulsifiers, antioxidants,sun screens, and a solvent or mixed solvent system Polymer thickenersthat may be used include those known to one skilled in the art, such ashydrophilic and hydroalcoholic gelling agents frequently used in thecosmetic and pharmaceutical industries. Other preferred gelling polymersinclude hydroxyethylcellulose, cellulose gum, MVE/MA decadienecrosspolymer, PVM/MA copolymer, or a combination thereof.

A composition comprising a lytic enzyme/polypeptide(s) and antibiotic(s)can be administered in the form of a candy, chewing gum, lozenge,troche, tablet, a powder, an aerosol, a liquid, a liquid spray, ortoothpaste for the prevention or treatment of bacterial infectionsassociated with upper respiratory tract illnesses. The lozenge, tablet,or gum into which the lytic enzyme/polypeptide(s) is added may containsugar, corn syrup, a variety of dyes, non-sugar sweeteners, flavorings,any binders, or combinations thereof. Similarly, any gum-based productsmay contain acacia, carnauba wax, citric acid, cornstarch, foodcolorings, flavorings, non-sugar sweeteners, gelatin, glucose, glycerin,gum base, shellac, sodium saccharin, sugar, water, white wax, cellulose,other binders, and combinations thereof. Lozenges may further containsucrose, cornstarch, acacia, gum tragacanth, anethole, linseed,oleoresin, mineral oil, and cellulose, other binders, and combinationsthereof. Sugar substitutes can also be used in place of dextrose,sucrose, or other sugars. Compositions comprising lytic enzymes, ortheir peptide fragments can be directed to the mucosal lining, where, inresidence, they kill colonizing disease bacteria. The mucosal lining, asdisclosed and described herein, includes, for example, the upper andlower respiratory tract, eye, buccal cavity, nose, rectum, vagina,periodontal pocket, intestines and colon. Due to natural eliminating orcleansing mechanisms of mucosal tissues, conventional dosage forms arenot retained at the application site for any significant length of time.

It may be advantageous to have materials which exhibit adhesion tomucosal tissues, to be administered with one or more phage enzymes andother complementary agents over a period of time. Materials havingcontrolled release capability are particularly desirable, and the use ofsustained release mucoadhesives has received a significant degree ofattention. Other approaches involving mucoadhesives which are thecombination of hydrophilic and hydrophobic materials, are known.Micelles and multilamillar micelles may also be used to control therelease of enzyme. Materials having capacity to target or adhere tosurfaces, such as plastic, membranes, devices utilized in clinicalpractice, including particularly any material or component which isplaced in the body and susceptible to bacterial adhesion or biofilmdevelopment, such as catheters, valves, prosthetic devices, drug orcompound pumps, stents, orthopedic materials, etc, may be combined,mixed, or fused to the lysin(s) of use in the present invention.

Therapeutic or pharmaceutical compositions of the invention can alsocontain polymeric mucoadhesives including a graft copolymer comprising ahydrophilic main chain and hydrophobic graft chains for controlledrelease of biologically active agents. The compositions of thisapplication may optionally contain other polymeric materials, such aspoly(acrylic acid), poly,-(vinyl pyrrolidone), and sodium carboxymethylcellulose plasticizers, and other pharmaceutically acceptable excipientsin amounts that do not cause deleterious effect upon mucoadhesivity ofthe composition.

A lytic enzyme/polypeptide(s) and antibiotic(s) of the invention may beadministered for use in accordance with the invention by anypharmaceutically applicable or acceptable means including topically,orally or parenterally. For example, the lytic enzyme/polypeptide(s) canbe administered intramuscularly, intrathecally, subdermally,subcutaneously, or intravenously to treat infections by gram-positivebacteria. In cases where parenteral injection is the chosen mode ofadministration, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are preferred. Stabilizers include gelatinand albumin. A vasoconstriction agent can be added to the formulation.The pharmaceutical preparations according to this application areprovided sterile and pyrogen free.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The effective dosage rates or amounts of the lytic enzyme/polypeptide(s)to be administered, and the duration of treatment will depend in part onthe seriousness of the infection, the weight of the patient,particularly human, the duration of exposure of the recipient to theinfectious bacteria, the number of square centimeters of skin or tissuewhich are infected, the depth of the infection, the seriousness of theinfection, and a variety of a number of other variables. The compositionmay be applied anywhere from once to several times a day or a week, andmay be applied for a short, such as days or up to several weeks, or longterm period, such as many weeks or up to months. The usage may last fordays or weeks. Any dosage form employed should provide for a minimumnumber of units for a minimum amount of time. The concentration of theactive units of enzymes believed to provide for an effective amount ordosage of enzymes may be selected as appropriate.

The lysin and antibiotics of use and application in the compositions andmethods of the invention may be administered simultaneously orsubsequently. The lysin and antibiotic agents may be administered in asingle dose or multiple doses, singly or in combination. The lysin andantibiotic may be administered by the same mode of administration or bydifferent modes of administration, and may be administered once, twiceor multiple times, one or more in combination or individually. Thus,lysin may be administered in an initial dose followed by a subsequentdose or doses, particularly depending on the response and bacterialkilling or decolonization, and may be combined or alternated withantibiotic dose(s). In a particular aspect of the invention and in viewof the reduction in the development of resistance to antibiotics byadministering a lysin, particularly PlySs2, with antibiotic,combinations of antibiotic and lysin may be administered for longerperiods and dosing can be extended without risk of resistance. Inaddition, in as much as the doses required for efficacy of each ofantibiotic and lysin are significantly reduced by combining orco-administering the agents simultaneously or in series, a patient canbe treated more aggressively and more continually without risk, or withreduced risk, of resistance.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds, addedadditional compound(s), or lysin enzyme compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term ‘assay’ means any process used to measure a specific propertyof a compound. A ‘screening assay’ means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

‘Therapeutically effective amount’ means that amount of a drug,compound, antimicrobial, antibody, polypeptide, or pharmaceutical agentthat will elicit the biological or medical response of a subject that isbeing sought by a medical doctor or other clinician. In particular, withregard to gram-positive bacterial infections and growth of gram-positivebacteria, the term “effective amount” is intended to include aneffective amount of a compound or agent that will bring about abiologically meaningful decrease in the amount of or extent of infectionof gram-positive bacteria, including having a bacteriocidal and/orbacteriostatic effect. The phrase “therapeutically effective amount” isused herein to mean an amount sufficient to prevent, and preferablyreduce by at least about 30 percent, more preferably by at least 50percent, most preferably by at least 90 percent, a clinicallysignificant change in the growth or amount of infectious bacteria, orother feature of pathology such as for example, elevated fever or whitecell count as may attend its presence and activity.

The term ‘treating’ or ‘treatment’ of any disease or infection refers,in one embodiment, to ameliorating the disease or infection (i.e.,arresting the disease or growth of the infectious agent or bacteria orreducing the manifestation, extent or severity of at least one of theclinical symptoms thereof). In another embodiment ‘treating’ or‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or infection,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, ‘treating’ or ‘treatment’ relates to slowingthe progression of a disease or reducing an infection.

It is noted that in the context of treatment methods which are carriedout in vivo or medical and clinical treatment methods in accordance withthe present application and claims, the term subject, patient orindividual is intended to refer to a human.

The terms “gram-positive bacteria”, “Gram-positive bacteria”,“gram-positive” and any variants not specifically listed, may be usedherein interchangeably, and as used throughout the present applicationand claims refer to Gram-positive bacteria which are known and/or can beidentified by the presence of certain cell wall and/or cell membranecharacteristics and/or by staining with Gram stain. Gram positivebacteria are known and can readily be identified and may be selectedfrom but are not limited to the genera Listeria, Staphylococcus,Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, andClostridium, and include any and all recognized or unrecognized speciesor strains thereof. In an aspect of the invention, the PlyS lysinsensitive gram-positive bacteria include bacteria selected from one ormore of Listeria, Staphylococcus, Streptococcus, and Enterococcus.

The term “bacteriocidal” refers to capable of killing bacterial cells.

The term “bacteriostatic” refers to capable of inhibiting bacterialgrowth, including inhibiting growing bacterial cells.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1

PlySs2 lysin demonstrates the ability to kill various strains ofclinically significant gram-positive bacteria, including antibioticresistant strains such as methicillin and vancomycin resistant andsensitive strains of Staphylococcus aureus (MRSA, MSSA, VRSA, VISA),daptomycin-resistant Staphylococcus aureus (DRSA), andlinezolid-resistant Staphylococcus aureus (LRSA). PlySs2 is a uniquelysin in having broad species killing activity and can kill multiplespecies of bacteria, particularly gram-positive bacteria, includingStaphylococcus, Streptococcus, Enterococcus and Listeria bacterialstrains. A tabulation of sensitivity (as depicted using MIC values anduM concentrations) of staphylococci to PlySs2 lysin and variousantibiotics is shown in TABLE 2. Minimally inhibitory concentration(MICs) were determined using the broth microdilution method inaccordance with standards and as described in the Clinical andLaboratory Standards Institute (CLSI) document M07-A9 (Methods fordilutional antimicrobial sensitivity tests for bacteria that growaerobically. Volume 32 (Wayne [PA]: Clinical and Laboratory StandardsInstitute [US], 2012). This value is the MIC determined in the presenceof reducing agent (such as DTT or BMS) in the MIC assay. Reducing agentis added for the purpose of improving reproducibility between and amongassays in determining MIC values.

TABLE 2

*MICs were determined using the broth microdilution method andevaluating 80% growth inhibition according to CLSI methods (M07-A9).

Activity of PlySs2 against various gram-positive and gram-negativeorganisms is tabulated in TABLE 3, which includes MIC values and rangefor the different organisms. Activity of PlySs2 againstantibiotic-resistant Staphylococcus aureus is provided in TABLE 4.PlySs2 has potent growth inhibitory activity on all Staphylococcusaureus strains tested including 103 MSSA and 120 MRSA isolates (MIC=8μg/mL) as well as Groups A and B streptococci and Staphylococcuslugdiensis (TABLE 3). Little or no activity was observed on a collectionof other Gram positive bacteria as well as all Gram negative bacteriatested. Although PlySs2 effectively kills antibiotic resistant andsensitive S. aureus as well as numerous other clinically significantgram-positive bacteria, it is notably ineffective on numerous commensalbacteria, such as Escherichia coli, as shown above in TABLE 1 and inTABLE 5 below which provides sensitivity of human gut bacteria andPlySs2 MIC.

TABLE 3 Activity of PlySs2 Against Gram-Positive and Gram-NegativeOrganisms Organism and susceptibility subset MIC (μg/mL) (no. tested)50% 90% Range Staphylcoccus aureus Methicillin susceptible (103) 4 81-16 Methicillin resistant (120) 4 8 1-16 Streptococcus pyogenes, GroupA (54) 2 8 0.5-8   Streptococcus agalactiae, Group B (51) 8 16 1-64Other Gram-positive organisms Staphylococcus lugdiensis (10) 8 8 8Staphylococcus epidermidis (11) 128 512  4-512 Streptococcus pneumoniae(26) 16 64 1-64 Streptococcus mutans (12) 64 256  2-256 Listeriamonocytogenes (12) 128 512  1-512 Enterococcus faecalis (17) >512 >512 32->512 Enterococcus faecium (5) >512 >512  32->512 Bacillus cereus(10) >512 >512 >512 Gram-negative organisms Acinetobacter baumannii(8) >512 >512 >512 Escherichia coli (6) >512 >512 >512 Pseudomonasaeruginosa (5) >512 >512 >512

TABLE 4 Activity of PlySs2 Against Antibiotic- Resistant Staphylococcusaureus MIC (mg/mL) Susceptibility subset (no. tested) 50% 90% RangeVancomycin-resistant (14) 2 4 1-4 Vancomycin-intermediate (31) 8 32 1-64 Linezolid-resistant (5) 2 2 2-4 Daptomycin-resistant (8) 2 4 2-4

TABLE 5 Sensitivity of Human Gut Bacteria to PlySs2 Organism N CF-301MIC (ug/ml) Salmonella enteriditis 1 >512 Pseudomonas aeruginosa 11 >512Escherichia coli 10 >512 Klebsiella spp. 8 >512 Proteus mirabilis 2 >512Lactobacillus spp. 6 >512 Lactococcus spp. 3 >512

While PlySs2 is effective against many different clinically relevantbacteria, it retains the beneficial character of many lysins in lackingbroad spectrum bacterial killing, therefore side effects such asintestinal flora disturbance seen with many antibiotics will beminimized. In addition, lysins have demonstrated a low probability ofbacterial resistance. PlySs2's remarkably broad clinically relevantkilling capability make it uniquely applicable to the clinical setting,including in instances where there is a fully uncharacterized or mixedbacterial infection.

Staphylococcus aureus is the causative agent of several seriousinfectious diseases and the emergence of antibiotic resistant S. aureusstrains has resulted in significant treatment difficulties, intensifyingthe need for new antimicrobial agents. Currently, 40 to 60% ofnosocomial infections of S. aureus are resistant to oxacillin (Massey RC et al (2006) Nat Rev Microbiol 4:953-958), and greater than 60% of theisolates are resistant to methicillin (Gill S R et al (2005) J Bacteriol187:2426-2438). A number of new antimicrobial agents, such as linezolid,quinupristin-dalfopristin, daptomycin, telavancin, new glycopeptides,ceftaroline, and ceftobiprole, have been introduced or are underclinical development (Aksoy D Y and S Unal (2008) Clin Microbiol Infect14:411-420). As an option, current antibiotics to which strains such asMRSA are resistant may be resurrected as viable candidates in thetreatment of MRSA when used in combination with other agents, offering anew dimension of potential anti-infectives. The application and use oflysin in combination with antibiotic has potential to circumventbacterial resistance by virtue of the very low probability ofdevelopment of resistance to the lysin component.

In order to more fully evaluate PlySs2's applicability to clinicalStaphylococcal infections, time kill studies were undertaken againstnumerous Staphylococcus aureus strains, including methicillin resistantand methicillin sensitive strains. Time kill assays were performedaccording to CLSI methodology (CLSI document M07-A9 column 32 No. 2) on42 methicillin resistant S. aureus (MRSA) strains and 20 methicillinsensitive S. aureus (MSSA) strains. Cultures of each strain(5.5×10⁵-1×10⁶ starting inoculum) were treated with PlySs2 lysin andwith antibiotics daptomycin, oxacillin or vancomycin for comparison for6 hours with aeration. MRSA and MSSA strains were treated with PlySs2,daptomycin and vancomycin. MSSA strains were also treated withoxacillin. 1× MIC concentrations of the different antibiotics wereutilized, based on published and established antibiotic MIC values.PlySs2 lysin treatment was at approximately 1× MIC as previouslydetermined (see TABLE 2 above). Culture aliquots were removed hourly upto 6 hours (time points taken at 15 and 30 min, 1 hr, 2 hr, 3 hr, 4 hr,5 hr, and 6 hr) and added to a PBS/charcoal solution (to inactivate eachdrug), which was then serially diluted and plated for bacterialviability. The resulting log CFU/mL was plotted for each culture. Growthcontrols were included for each strain and represent bacterial growth inthe absence of any antibacterial agent. Exemplary log kill curves forselected MRSA strains are depicted in FIG. 1. Exemplary log kill curvesfor selected MSSA strains are depicted in FIG. 2. A summary plot of thetime kill studies with the MRSA and MSSA strains is shown in FIG. 3.

A listing of strains used in the studies provided herein, includingcross-reference for recognized and available strain names is providedbelow in TABLE 6.

TABLE 6 Strain List Laboratory Designation Strain Common Strain (CFS #)Type Designation* 223 MRSA BAA-1720 241 MRSA NRS100 243 MSSA NRS107 245MRSA NRS070 250 MSSA NRS149 253 MSSA NRS155 254 MSSA NRS156 263 MRSANRS387 269 MRSA NRS123 (MW2) 270 MRSA NRS383 553 MRSA ATCC 43300 554MSSA ATCC 25923 581 MSSA ATCC 29213 650 MRSA 052C 738 MRSA NRS192 743MRSA NRS255 832 MRSA NRS671 926 MRSA BK20781 927 MRSA W15 *NARSA (“NRS”)and ATCC (“ATCC” and “BAA”) strain designations are indicated whereappropriate. Additional names reflect strain designations available inthe literature.

PlySs2 has Rapid Kill Kinetics in vitro

Rapid-kill kinetics are desirable in the clinical setting to treatpatients with fulminant bacterial infections. To test the rate ofantimicrobial activity in vitro, we used time-kill assays (Mueller M etal (2004) Antimicrob Agents Chemotherapy 48:369-377) in which 1× MICdrug concentrations were tested across 20 different MSSA and 42 MRSAstrains. PlySs2 reached bactericidal levels (Methods for dilutionantimicrobial susceptibility tests for bacteria that grow aerobically.Vol. 32 (Wayne (Pa.): Clinical and Laboratory Standards Institute(U.S.), 2012) (≥3-log₁₀ reduction in CFUs) within 30 minutes (FIGS. 4Aand B). In contrast, daptomycin required 6 hours to reach bactericidallevels while vancomycin and oxacillin achieved only 2-log₁₀ kill within6 hours. Rapid-kill kinetics were also obtained for PlySs2 against setsof 15 different contemporary MSSA (FIG. 4C) or MRSA (FIG. 4D) isolates,illustrating the efficient bactericidal activity of PlySs2 on relevantclinical isolates. The potent activity of PlySs2 was further illustratedby electron microscopy showing extensive bacteriolysis of S. aureuscocci after only 15 seconds of treatment; the speed of PlySs2 action isconsistent with a bactericidal effect immediately upon contact (FIG.4E).

All MRSA and MSSA strains tested are killed rapidly with PlySs2, withmaximal kill (ie, ≥3 log reduction) observed generally within the firsthour of incubation with lysin and logs reduced to 1 log CFU/ml (theeffective lower limit of the test) in most instances. Daptomycin orvancomycin reduce growth of most MRSA and MSSA strains by 2-3 logsobserved generally over a few hours or more of incubation, withdaptomycin being the most effective against most strains. Oxacillin wasthe least effective of the antibiotic agents against MSSA strains. Inall instances, PlySs2 kill was greater and faster than any antibiotic.

The studies were expanded to include testing of various S. aureusstrains, including vancomycin intermediate sensitive S. aureus (VISA),vancomycin resistant S. aureus (VRSA), linezolid resistant S. aureus(LRSA) and daptomycin resistant S. aureus (DRSA), with PlySs2 lysin,daptomycin, vancomycin and linezolid, using methods as described above.A tabulation of studies undertaken with MSSA, MRSA, VISA, VRSA, LRSA andDRSA strains of Staphylococcus aureus is provided in TABLE 2 above withminimal inhibitory concentrations (MICs) of PlySs2 and variousantibodies provided for various strains.

Example 2

While PlySs2 lysin alone kills more rapidly than antibiotics alone, asshown above, no information regarding the capability or effectiveness ofPlySs2 in combination with antibiotics is known or available. Bacterialkill studies were undertaken to assess combinations of PlySs2 lysin withantibiotic against Staphylococcus aureus in vitro.

Time kill assays were performed as described above on several MRSAstrains with addition of PlySs2 or antibiotic alone or in combination atvarious concentrations. Cultures of each strain (5.5×10⁵-1×10⁶ startinginoculum) were treated with PlySs2 lysin, antibiotic (daptomycin orvancomycin), or combinations of PlySs2 and antibiotic for 6 hours withaeration. In each instance, sub-MIC doses of PlySs2 and of antibioticwere utilized in order to observe synergy and enhanced combination agenteffectiveness. Growth controls were included for each strain andrepresent bacterial growth in the absence of any antibacterial agent.Time kill curves of two MRSA strains with addition of ½ MIC of PlySs2and ¼ MIC of vancomycin are shown in FIG. 5. At these sub-MIC doses,vancomycin or PlySs2 are ineffective or poorly effective alone up to 6hours. Combinations of ½ MIC of PlySs2 and ¼ MIC of vancomycin result inup to 4 logs of kill of MRSA in culture within 6 hours.

Log Kill curves of two MRSA strains with addition of ¼ MIC of PlySs2 and⅛ MIC of daptomycin are shown in FIG. 6. Combinations of ¼ MIC of PlySs2and ⅛ MIC of daptomycin result in approximately 4 logs of kill of MRSAin culture within 6 hours. FIG. 7 depicts another combination studybased on 1× MIC values of PlySs2 and daptomycin on MRSA strain 650 (O52CTomasz strain-1×10⁹ starting inoculum). PlySs2 lysin is added at 16μg/ml, daptomycin is added at 1 μg/ml. While each single agent aloneresults in 4-5 log kill at the added concentrations, the combination ofPlySs2 and daptomycin provides complete kill (log kill to the detectionlimit of the assay) within 2 hours.

Example 3

Combination therapy is particularly effective when drugs actsynergistically (Cottarel G & Wierzbowski J (2007) Trends Biotechnology25:547-555). Synergy assessment between PlySs2 and cell envelope-activeantibiotics was performed by the time-kill assay, a preferred method forexamining synergistic antimicrobial activity in vitro (Mueller M et al(2004) Antimicrob Agents and Chemotherapy 48:369-377; Methods fordilution antimicrobial susceptibility tests for bacteria that growaerobically, Vol. 32 (Wayne (Pa.): Clinical and Laboratory StandardsInstitute (U.S.), 2012). Synergy studies were additionally evaluatedwith antibiotic oxacillin, which is a member of the penicillin familyand kills bacteria in a distinct manner versus either of vancomycin ordaptomycin. The results of oxacillin studies either alone or in thepresence of lysin PlySs2 are shown in FIG. 8. Time-kill curves weregenerated using sub-MIC concentrations of PlySs2 daptomycin, vancomycin,and oxacillin either alone or in combinations against clinical MRSA(FIG. 8C-8F) or MSSA (FIG. 8A-8B) isolates. Synergy was defined as a≥2-log10 decrease in CFU/mL at the 6 hour time-point for the combinationcompared to the most active single-agent. Based on this criteria, PlySs2acted synergistically with all antibiotics evaluated against allrepresentative MRSA and MSSA strains evaluated (see FIGS. 4-8). Anexpanded set of isolates were similarly examined and synergy wasobserved in 45 of 49 analyses for MSSA and 24 of 26 for MRSA with PlySs2combined with distinct antibiotics, including oxacillin, vancomycin anddaptomycin (TABLES 7-11 provided below).

TABLE 7 Synergy Time-Kill Results with Oxacillin (MSSA) OptimalSynergistic Concentrations² [PlySs2] [Oxacillin] ΔLog₁₀ Inter- Strains¹μg/mL (xMIC⁵) μg/mL (xMIC) CFU/mL³ action⁴ ATCC 25923 4.0 (0.13) 0.06(0.5) 2.9 Synergy ATCC 29213 1.0 (0.13) 0.13 (0.5) 2.3 Synergy JMI 12591.0 (0.13) 0.13 (0.5) 2.6 Synergy JMI 1787 0.5 (0.06) 0.13 (0.5) 4.0Synergy JMI 6408 1.0 (0.13) 0.10 (0.4) 3.3 Synergy JMI 6686 0.5 (0.06)0.13 (0.5) 4.0 Synergy JMI 7140 0.5 (0.13) 0.50 (0.5) 4.5 Synergy JMI8928 1.0 (0.13) 0.19 (0.4) 2.9 Synergy JMI 9365 0.3 (0.06) 0.13 (0.5)2.5 Synergy JMI 11146 0.3 (0.03) 0.13 (0.5) 2.9 Synergy JMI 13734 0.5(0.13) 0.10 (0.4) 4.0 Synergy JMI 13736 0.5 (0.13) 0.10 (0.4) 0.8Additive JMI 15395 1.0 (0.06) 0.13 (0.5) 3.3 Synergy JMI 16140 2.0(0.13) 0.10 (0.4) 3.0 Synergy JMI 33611 0.5 (0.06) 0.25 (0.5) 3.7Synergy JMI 40979 0.5 (0.13) 0.25 (0.5) 3.0 Synergy Legend for TABLES7-11: ¹ATCC quality control strains and JMI isolate numbers are shown.²Concentrations of PlySs2 and antibiotic used in synergy time-killexperiments. Values were carefully determined in range-finding studiesand represent concentrations that most closely approach ideal levels ofPlySs2 (that is, resulting in a ~2-log₁₀ decrease in viability comparedto growth control) and antibiotic (that is, resulting in <1 log decreasein viability compared to growth control). ³Decreases in log₁₀ colonycounts (or ΔLog₁₀ CFU/mL) are shown for cultures treated for 6 hourswith drug combination, compared to cultures treated with the most activesingle agent. ⁴Synergy is defined by the CLSI²¹ as a ≥2-log₁₀ decreasein CFU/mL. Additive interactions are defined as a <2-log₁₀ decrease inCFU/mL. ⁵xMIC, denotes percentage of MIC represented by eachconcentration. For example, an xMIC value of 0.5 means that the optimalsynergistic concentration for a particular drug is ½ the specific MICvalue of a particular isolate or strain. The xMIC value is, therefore,the concentration of drug used in synergy time-kill assay divided by theMIC for that drug against the specific strain in the absence ofreductant. Key: ΔLog₁₀ CFU/mL = change in log₁₀ colony-forming units;MIC = minimum inhibitory concentration.

TABLE 8 Synergy Time-Kill Results with Vancomycin (MSSA) OptimalSynergistic Concentrations² PlySs2 Vancomycin ΔLog₁₀ Inter- Strains¹μg/mL (xMIC⁵) μg/mL (xMIC) CFU/mL³ action⁴ ATCC 29213 1.0 (0.03) 0.5(0.5) 2.3 Synergy JMI 1259 1.0 (0.13) 0.5 (0.5) 3.5 Synergy JMI 1787 1.0(0.13) 0.5 (0.5) 3.1 Synergy JMI 6408 0.5 (0.06) 0.5 (0.5) 2.8 SynergyJMI 6686 1.0 (0.13) 0.5 (0.5) 5.0 Synergy JMI 7140 0.5 (0.13) 0.5 (0.5)3.3 Synergy JMI 8928 0.5 (0.06) 0.5 (0.5) 1.8 Additive JMI 9365 0.5(0.13) 0.5 (0.5) 2.6 Synergy JMI 11146 0.5 (0.06) 0.3 (0.5) 3.3 SynergyJMI 13734 0.5 (0.13) 0.5 (0.5) 4.3 Synergy JMI 13736 0.5 (0.13) 0.3(0.3) 2.3 Synergy JMI 15395 0.5 (0.03) 0.5 (0.5) 3.0 Synergy JMI 161401.0 (0.06) 0.5 (0.5) 3.3 Synergy JMI 18219 0.5 (0.13) 0.5 (0.5) 3.4Synergy JMI 33611 0.5 (0.06) 0.5 (0.5) 3.6 Synergy JMI 40979 0.5 (0.13)0.5 (0.5) 3.8 Synergy

TABLE 9 Synergy Time-Kill Results with Daptomycin (MSSA) OptimalSynergistic Concentrations² PlySs2 Daptomycin ΔLog₁₀ Inter- Strains¹μg/mL (xMIC⁵) μg/mL (xMIC) CFU/mL³ action⁴ ATCC 25923 0.5 (0.02) 0.25(0.50) 3.1 Synergy ATCC 29213 0.3 (0.03) 0.25 (0.50) 4.3 Synergy JMI1259 1.0 (0.13) 0.13 (0.25) 3.5 Synergy JMI 1787 0.5 (0.06) 0.07 (0.14)3.0 Synergy JMI 6408 0.5 (0.06) 0.13 (0.25) 2.7 Synergy JMI 6686 1.0(0.13) 0.14 (0.28) 3.4 Synergy JMI 7140 0.5 (0.13) 0.13 (0.25) 2.6Synergy JMI 8928 0.5 (0.06) 0.13 (0.25) 2.9 Synergy JMI 9365 0.3 (0.06)0.07 (0.28) 1.8 Additive JMI 11146 0.5 (0.06) 0.13 (0.50) 4.2 SynergyJMI 13734 0.3 (0.06) 0.08 (0.17) 3.2 Synergy JMI 13736 0.5 (0.13) 0.19(0.38) 4.5 Synergy JMI 15395 1.0 (0.06) 0.25 (0.50) 3.2 Synergy JMI16140 0.5 (0.03) 0.13 (0.50) 1.9 Additive JMI 18219 0.5 (0.13) 0.13(0.25) 3.7 Synergy JMI 33611 0.3 (0.03) 0.13 (0.25) 3.4 Synergy JMI40979 0.3 (0.06) 0.08 (0.16) 2.5 Synergy

TABLE 10 Synergy Time-Kill Results with Vancomycin (MRSA) OptimalSynergistic Concentrations² PlySs2 Vancomycin ΔLog₁₀ Inter- Strains¹μg/mL (xMIC⁵) μg/mL (xMIC) CFU/mL³ action⁴ ATCC 43300 1.0 (0.13) 0.5(0.5) 2.1 Synergy JMI 2290 0.5 (0.06) 0.5 (0.5) 2.3 Synergy JMI 3345 0.5(0.13) 0.5 (0.5) 5.0 Synergy JMI 4564 0.5 (0.03) 0.5 (0.5) 2.7 SynergyJMI 4789 1.0 (0.13) 0.5 (0.5) 3.3 Synergy JMI 5506 0.5 (0.13) 0.3 (0.5)3.1 Synergy JMI 5675 0.5 (0.13) 0.5 (0.5) 3.2 Synergy JMI 6546 0.3(0.03) 0.5 (0.5) 2.6 Synergy JMI 8941 0.5 (0.03) 0.5 (0.5) 1.8 AdditiveJMI 12568 0.5 (0.13) 0.5 (0.5) 5.0 Synergy JMI 18233 0.5 (0.06) 0.5(0.5) 3.2 Synergy JMI 37753 0.5 (0.13) 0.5 (0.5) 1.6 Additive JMI 390860.5 (0.13) 0.3 (0.5) 2.5 Synergy

TABLE 11 Synergy Time-Kill Results with Daptomycin (MRSA) OptimalSynergistic Concentrations² PlySs2 Daptomycin ΔLog₁₀ Inter- Strains¹μg/mL (xMIC⁵) μg/mL (xMIC) CFU/mL³ action⁴ ATCC 43300 0.5 (0.06) 0.13(0.25) 4.1 Synergy JMI 2290 1.0 (0.13) 0.25 (0.25) 4.1 Synergy JMI 33451.0 (0.25) 0.25 (0.50) 5.0 Synergy JMI 4564 0.5 (0.03) 0.13 (0.25) 2.8Synergy JMI 4789 1.0 (0.13) 0.13 (0.25) 2.4 Synergy JMI 5506 0.5 (0.13)0.13 (0.25) 2.3 Synergy JMI 5675 0.5 (0.13) 0.13 (0.25) 3.4 Synergy JMI6546 1.0 (0.13) 0.06 (0.13) 3.3 Synergy JMI 8941 0.5 (0.06) 0.13 (0.25)2.1 Synergy JMI 12568 0.5 (0.13) 0.13 (0.50) 3.6 Synergy JMI 18233 1.0(0.13) 0.25 (0.25) 5.7 Synergy JMI 37753 0.5 (0.06) 0.13 (0.25) 2.3Synergy JMI 39086 0.5 (0.13) 0.13 (0.25) 3.5 Synergy

Example 4

Broth microdilution MIC testing was performed using 96 well panelsaccording to the methods described above for Example 1 (CLSImethodology, CLSI document M07-A9, column 32 no 2). In the presentstudies, however, both PlySs2 lysin and antibiotic daptomycin arepresent together in each well at different starting concentrations.Studies were completed with 12 different MRSA S. aureus strains. In eachinstance, the MIC of PlySs2 for the strain was first determined. DAPMICs for each strain were based on broth microdilution MIC testingaccording to published methodology and confirmed with published andavailable data for the tested isolates. 5.5×10⁵-1×10⁶ cells were addedto each well and grown in the presence of various amounts of PlySs2lysin and daptomycin for 24 hours at 37° C. without aeration. MIC valueswere determined by eye and confirmed by plating bacteria to determineviable cell counts in each well of a 96-well plate. Cultures wereassessed in the presence and absence of a reducing agent (e.g., betamercaptoethanol (BME), dithiothreitol (DTT)). MIC values are lowerrelatively in the presence of reducing agent and repeatability isimproved with reducing agent.

Dual Agent MIC Determinations.

The dual agent MIC assay is derived from the standard brothmicrodilution method (Methods for dilution antimicrobial susceptibilitytests for bacteria that grow aerobically (2012) Vol. 32 (Clinical andLaboratory Standards Institute (U.S.), Wayne (Pa.)). Whereas the MICassay dilutes one drug across the x-axis, the MIC combo assays dilutestwo drugs together across the x axis. Two to four 96-well polypropylenemicrotiter plates (Becton, Dickenson, and Company) are combined to yielddesired dilution schemes. PlySs2 is first diluted two-fold verticallydownward in column 1, yielding a concentration range of 2,048 to 1μg/mL. Daptomycin is next added at a constant concentration (4 μg/mL) toeach well of column 1. All of the wells of column 1, therefore,containing a dilution range of PlySs2 against a background of 4 μg/mLdaptomycin. Column 1 is next diluted two-fold across the entire x-axissuch that all the wells of column 11 have a daptomycin concentration of0.0037 μg/mL. After drug dilutions, cells are added (˜5×10⁵ CFUs/mL) andafter 24 hours of incubation at 35° C. in ambient air the MICs wasrecorded as the most dilute drug concentrations inhibiting bacterialgrowth.

Exemplary results with eight MRSA strains are depicted in FIGS. 9-18. Ineach of FIGS. 9-18, a twelve-well doubling dilution range proceeds tothe right for each panel row. Each panel represents the well of a96-well plate. The indicted bacterial strains were then inoculated intoeach well and incubated for 24 hours at which time growth was examined.Unshaded panels indicate wells in which drug combinations inhibitedgrowth. The yellow (light)-shaded panels indicate the lowestdrug-combination concentrations that inhibited growth (essentiallycorresponding to the MIC). The red (dark)-shaded panels indicatebacterial growth (in other words agent combinations that did not inhibitgrowth). Studies were conducted in the presence and absence of reducingagent. In each instance, with or without the reducing agent, significantsynergy was observed, with multi-fold reduction in amount of both lysinand antibiotic required when both were provided in combination.Reduction in antibiotic required was particularly significant with addedlysin.

The overall experimental results with a dozen MRSA strains aresummarized in TABLE 12 below. As shown below and depicted in FIGS. 9-18,combining PlySs2 and antibiotic (daptomycin) together cansynergistically achieve 2-4 fold reduction in the effective MIC ofPlySs2. Remarkably, combining PlySs2 and daptomycin together cansynergistically achieve 16-256 increased sensitivity (fold reduction) inthe effective MIC of the antibiotic daptomycin.

TABLE 12 PlySs2 Daptomycin MRSA MIC MIC Fold MIC MIC Fold STRAIN alone¹combo² reduction³ alone combo reduction 553 2 1 2 1 0.0075 128 223 2 0.54 1 0.0075 128 270 8 2 4 1 0.015 64 269 (MW2) 8 2 4 1 0.015 64 241 4 2 21 0.0037 256 263 8 2 4 1 0.0075 128 650 8 4 2 1 0.015 64 827 8 4 2 10.0075 128 828 8 2 4 1 0.0075 128 829 8 4 2 1 0.0075 128 830 4 2 2 10.015 64 833 8 2 4 1 0.0075 128 ¹The “MIC alone” value is thesingle-agent MIC (in μg/mL) for each drug. ²The “MIC combo” value is themost dilute concentration of each agent (in μg/mL) that, when combined,inhibited growth. ³Fold Reduction (Increased sensitivity) corresponds tothe MIC combo/MIC alone for each agent.

Example 5

Further assessment of synergy was undertaken by performing checkerboardassays and calculating fractional inhibitory concentration index (FICI)values (Tallarida R J (2012) J Pharmacol and Exper Therapeutics342:2-8). These studies were performed using exemplary antibioticsdaptomycin, vancomycin and oxacillin. Using this assessment, synergy isdefined as inhibitory activity greater than would be predicted by addingthe two drugs together (FICI of ≤0.5). Representative isobolograms forthe different antibiotics against MRSA and MSSA strains are provided inFIG. 19. Synergy was observed for 79% (daptomycin), 86% (vancomycin),and 100% (oxacillin) of the 29 MSSA strains and for 89% (daptomycin) and69% (vancomycin) of the 26 MRSA strains. The results are tabulated belowin TABLES 13-15.

TABLE 13 Checkerboard Analyses of PlySs2 Combined with Oxacillin,Vancomycin, or Daptomycin (MSSA) Oxacillin Vancomycin Daptomycin Inter-Inter- Inter- Strains FIC_(min) action FIC_(min) action FIC_(min) actionATCC 0.156 Synergy 0.500 Synergy 0.500 Synergy 25923 ATCC 0.250 Synergy0.500 Synergy 0.750 Additive 29213 JMI 243 0.187 Synergy 0.375 Synergy0.312 Synergy JMI 332 0.187 Synergy 0.281 Synergy 0.312 Synergy JMI 11040.375 Synergy 0.375 Synergy 0.265 Synergy JMI 1259 0.187 Synergy 0.500Synergy 0.500 Synergy JMI 1282 0.375 Synergy 1.00 Additive 0.750Additive JMI 1787 0.375 Synergy 0.500 Synergy 0.562 Additive JMI 35210.250 Synergy 0.75 Additive 0.375 Synergy JMI 3671 0.312 Synergy 0.562Additive 0.500 Synergy JMI 4811 0.375 Synergy 0.375 Synergy 0.500Synergy JMI 6408 0.375 Synergy 0.375 Synergy 0.312 Synergy JMI 64140.375 Synergy 0.375 Synergy 0.375 Synergy JMI 6544 0.250 Synergy 0.500Synergy 0.375 Synergy JMI 6686 0.281 Synergy 0.500 Synergy 0.375 SynergyJMI 7140 0.375 Synergy 0.375 Synergy 0.500 Synergy JMI 8928 0.375Synergy 0.375 Synergy 0.500 Synergy JMI 9365 0.125 Synergy 0.375 Synergy0.500 Synergy JMI 11146 0.250 Synergy 0.375 Synergy 0.375 Synergy JMI13734 0.375 Synergy 0.500 Synergy 0.375 Synergy JMI 13736 0.281 Synergy0.500 Synergy 0.500 Synergy JMI 15395 0.312 Synergy 0.250 Synergy 0.500Synergy JMI 16195 0.375 Synergy 0.375 Synergy 0.281 Synergy JMI 161400.375 Synergy 0.375 Synergy 0.531 Additive JMI 18219 0.500 Synergy 0.500Synergy 0.562 Additive JMI 24368 0.375 Synergy 0.375 Synergy 0.375Synergy JMI 29793 0.375 Synergy 0.56 Additive 0.562 Additive JMI 336110.312 Synergy 0.375 Synergy 0.375 Synergy JMI 40979 0.312 Synergy 0.375Synergy 0.312 Synergy Key: FIC_(min) = minimum fractional inhibitoryconcentration

TABLE 14 Checkerboard Analyses of PlySs2 Combined with Vancomycin orDaptomycin (MRSA) Vancomycin Daptomycin Strains FIC_(min) InteractionFIC_(min) Interaction ATCC 43300 0.500 Synergy 0.5 Synergy JMI 12250.625 Additive 0.375 Synergy JMI 1280 0.375 Synergy 0.375 Synergy JMI2290 0.500 Synergy 0.500 Synergy JMI 3345 0.375 Synergy 0.375 SynergyJMI 3346 0.375 Synergy 0.375 Synergy JMI 4564 0.375 Synergy 0.500Synergy JMI 4789 0.750 Additive 0.560 Additive JMI 5506 0.562 Additive0.375 Synergy JMI 5675 0.375 Synergy 0.375 Synergy JMI 6378 0.500Synergy 0.560 Additive JMI 6182 1.060 Additive 0.500 Synergy JMI 65460.375 Synergy 0.375 Synergy JMI 7053 0.375 Synergy 0.500 Synergy JMI8941 0.375 Synergy 0.500 Synergy JMI 9328 0.562 Additive 0.375 SynergyJMI 10339 0.531 Additive 0.500 Synergy JMI 11127 0.531 Additive 0.625Additive JMI 12568 0.250 Synergy 0.375 Synergy JMI 15992 0.187 Synergy0.500 Synergy JMI 18233 0.375 Synergy 0.500 Synergy JMI 37753 0.375Synergy 0.375 Synergy JMI 39086 0.500 Synergy 0.500 Synergy JMI 398480.500 Synergy 0.375 Synergy JMI 43255 0.375 Synergy 0.375 Synergy JMI44465 0.625 Additive 0.500 Synergy Key: FIC_(min) = minimum fractionalinhibitory concentration.

TABLE 15 Summary of PlySs2Interactions with Antimicrobial Agents Basedon Checkerboard Assays and Calculated FIC Values % Interactions withPlySs2^(a)) Oxacillin Vancomycin Daptomycin Species (N) SynergisticAdditive Synergistic Additive Synergistic Additive MSSA (29) 100 0 86.213.8 79.3 20.7 MRSA (26) NA NA 69.2 30.8 88.5 11.5 Key: FIC = fractionalinhibitory concentration; NA = not applicable.In the checkerboard assay, drug interactions are defined as eithersynergistic, additive, or antagonistic based on the FIC_(min) for eachcombination. The FIC for a drug is defined as the MIC of the drug incombination divided by the MIC of the drug used alone. The FIC_(min) isbased on the sum of FICs for each drug. If the FIC_(min) is ≤0.5, thecombination is interpreted as being synergistic; between >0.5 and ≤2 asadditive; and >2 as antagonistic.

Example 6 PlvSs2 Accelerates Antibiotic Binding to the Cell Envelope

As a complement to the synergy studies, daptomycin and vancomycin cellenvelope-binding was examined using BODIPY-fluorescein-labeledantibiotics in the presence and absence of sub-MIC levels of CF-301. Atime-course analysis of daptomycin binding (FIG. 20A) shows antibioticpenetration after only 15 minutes in the presence of CF-301 (at1/32^(nd) MIC) versus 3 hours without CF-301. Similarly, cellwall-labeling with vancomycin occurs within 5 minutes in the presence ofCF-301 (⅛^(th) MIC) versus 30 minutes without CF-301 (FIG. 20B). Forboth antibiotics, the labeling was first observed at bacterial divisionplanes.

Example 7

Daptomycin binds avidly to pulmonary surfactant and therefore is noteffective in treatment of staphylococcal pneumonia. In view of theeffectiveness of PlySs2 and daptomycin in combination againstsusceptible bacteria, a shown above, PlySs2 lysin and daptomycin wereevaluated alone and in combination in the presence of a commerciallyavailable surfactant, to mimic pulmonary surfactant.

MRSA strain MW2 and MSSA strain ATCC 29213 were used in these studies.Daptomycin and PlySs2 lysin were first evaluated alone in the presenceof surfactant (Survanta, Abbott Laboratories). The MICs of daptomycinand PlySs2 for each strain were determined in the presence of Survantausing broth microdilution methods. Doubling-dilution series wereestablished in the presence and absence of surfactant at concentrationsranging from 0% up to 15%. MICs were scored by eye at 24 hours andconfirmed by CFU counts in all wells. A similar study is reported bySilverman et al., 2005 (JID, volume 191, 2149-52) using MSSA strain 581.The fold change in MIC for daptomycin and CF301 at each surfactantconcentration (compared to MICs obtained in the absence of surfactant)were then calculated. The fold change in MIC at surfactantconcentrations for each strain is depicted in FIG. 21. In the presenceof Survanta as surfactant, daptomycin MIC is inhibited up to 256 fold.At a surfactant concentration of 1.25%, daptomycin is inhibited morethan 20 fold. In contrast, the MIC of PlySs2 is inhibited 8 fold,consistently across a range from 1.25 to 15% surfactant.

The effects of combinations of PlySs2 lysin and daptomycin wereevaluated in the presence of 15% surfactant (Survanta) in a combinationMIC study. The experimental setup is similar to that described above forthe combination MIC studies without surfactant (see Example 3). Theresults of synergy evaluation in the presence of surfactant are shown inFIG. 22. Briefly, the PlySs2 lysin plus daptomycin concentrations shownin the left-most wells were diluted two-fold across all twelve wells ofeach row. MRSA strain 269 (MW2) cells (5.5×10⁵-1×10⁶) were then added toeach well and incubated for 24 hours before growth was assessed.Unshaded wells indicate growth inhibition. Yellow (lightly)-shaded wellsindicate the most dilute drug combinations that still inhibit growth(ie, the MIC). Red (dark)-shaded wells indicate drug combinations thatallow growth. The PlySs2 synergy dose is 2 μg/ml or ⅛ of the MIC for thestrain tested. In this study, daptomycin is effective to 0.25 μg/ml,which corresponds to 1/1024 MIC of daptomycin.

Example 8 Combination Versus Single-Agent Therapy in Murine Models ofBacteremia

Animal studies were undertaken to assess the effect of PlySs2 incombination with antibiotic against S. aureus infection in vivo inmurine models of bacteremia. BALB/c mice were injected IP with differentlevels inoculums of MRSA strains and the animals are then dosed withdrug—either antibiotic, PlySs2 lysin, or a combination of antibiotic andPlySs2.

In a first set of studies using inoculums in the range of 10⁶ ofbacteria, 35 ug of daptomycin was injected subcutaneously (sc) at 5 hrspost bacterial infection, in a single dose. This dose is equivalent to1.75 mg/kg dose of daptomycin for a 20 gram mouse, while the humanequivalent dose of daptomycin in mice would be 50 mg/kg. Dosing of 1.75mg/kg of daptomycin in mice is equivalent to about 3.5% of the humanequivalent dose. PlySs2 was injected IP three times a day (TID), with 15μg of PlySs2 administered at 5 hrs, 9 hrs, and 13 hrs post bacterialinfection (15 μg is approximately equal to a dose of 0.8 mg/kg for a 20gram mouse). The animals are monitored and percent survival recordedevery three hours up to 24 hours post infection. Animal survival withMRSA (strain 269 or MW2) doses of 1.8×10⁶, 1.1×10⁶, 3.0×10⁶ and 3.1×10⁶bacteria was assessed and a compiled graph of survival data is providedin FIG. 23 for this MRSA strain. Similar studies were conducted withother S. aureus strains (strains 220 and 833) with comparable results(FIGS. 24-26). In all instances, animal survival was remarkably enhancedby combination dosing with PlySs2 lysin and antibiotic daptomycin. Thesestudies provide in vivo evidence of the efficacy of combination therapyof PlySs2 lysin with antibiotic daptomycin compared to PlySs2 lysin orantibiotic daptomycin alone.

High-Dose Inoculum Studies

Additional animal studies in a mouse bacteremia model were conducted toassess the ability of PlySs2 to enhance the efficacy of standard-of-careantibiotics in vivo. In the low challenge model (up to 7×10⁶ CFUinoculum with dosing at 4 hours), single-agent therapy administered as asingle dose of either 1.25 mg/kg PlySs2 or 2 mg/kg daptomycin resultedin 13% and 23% survival at 72 hours, respectively (FIG. 27A). Uponcombination of PlySs2 with daptomycin a significant enhancement wasobserved with a 72 hour survival rate of 73% (P<0.0001). The combinationof PlySs2 with daptomycin was therefore superior to each agent aloneunder low challenge conditions.

To test how robust the PlySs2 combination therapy may be, we increasedthe bacterial inoculum to a point where human-simulated doses ofsingle-agent antibiotics were poorly efficacious. In this high challengemodel (10⁹ CFU inoculum with dosing at 2 hours), human-simulated dosesof either daptomycin (50 mg/kg once daily)²⁶ or vancomycin (110 mg/kgtwice daily)²⁷ as single agents yielded 24 hour survival rates of 47%and 20%, respectively, and 72 hour survival rates of 31% and 3%,respectively (FIGS. 27B and 27E). PlySs2 administered as a single agentsimilarly yielded survival rates of 56/60% and 18/3% at 24 and 72 hours,respectively. In contrast, PlySs2 in combination with either daptomycinor vancomycin achieved 24 hour survival rates of 87% and 93% at 24 hoursand 82% and 67% at 72 hours, respectively, demonstrating superiority ofthe combination therapies over single-agent regimens under thesechallenging infection conditions. Additional PlySs2/daptomycincombination experiments were performed with two additional MRSA strains,yielding similar results (FIGS. 27C and 27D). When PlySs2 was furthertested in combination with oxacillin using a MSSA strain as theinoculum, the combination treatment was again superior to that of thesingle agents (FIG. 27F). Taken together, the results demonstrate thatPlySs2/antibiotic combinations are more efficacious than single-agentregimens for treating bacteremia and that statistically significantresults are obtained across various standard-of-care antibiotics andacross multiple S. aureus strains (P<0.0001 in all cases).

Lysin PlySs2 Demonstrates Dose-Responsive, Rapid-Kill Kinetics in-vivo

In a murine bacteremia model, PlySs2 exhibits a clear dose-response withsurvival enhancement over that observed for the mock-injected controlwith as little as 0.25 mg/kg and significant protection at the 5 mg/kgdose (data not shown). To assess the speed of bactericidal activity ofPlySs2 in vivo, MRSA CFUs were measured in the blood of infected micebefore and after administration of PlySs2. Upon dosing 5.25 mg/kg PlySs2at 2-hours post-infection, a 0.5-log₁₀ decrease in CFUs occurred in 15minutes and a 2-log₁₀ log decrease was observed within the 60 minutes oftreatment, demonstrating the rapid bactericidal activity of PlySs2 inthe bloodstream of infected animals (data not shown).

Murine Bacteremia Model.

Female BALB/c (inbred strain) mice, 5-7 weeks of age, 16.0-19.5 g bodyweight were purchased from Jackson Laboratories, Bar Harbor, Me. andutilized in all mouse experiments. Exponential phase bacterial inoculawere generated by allowing bacterial cells to grow to an optical densityof ˜0.5 at 600 nm, harvested, washed, and concentrated between1.5×10⁷-2×10⁹ CFU/ml. The bacterial pellet was suspended in anappropriate volume of 5% (w/v) mucin (Sigma Lot# SLBD5666V or SLBD5666V)to achieve the specific inoculum and placed on wet ice. Five hundred μL(˜7.5×10⁶-1×10⁹ CFU) were injected i.p. into mice. The study drug doseswere weight-adjusted with the injected volume between 160-200 μL.Post-infection survival was assessed every 3 or 6 hours for the first 24hours, then at 48 and 72 hours. The experiments were repeated 2-3 timeswith each treatment group containing between 10-20 mice. Allexperimental manipulations using the infectious agent were conducted ina BSL-2 hood. Dead animals were removed upon observation of mortality.

Example 9

Serial passage experiments were conducted with MRSA strain ATCC 700699and MSSA strain ATCC 25923 to generate and evaluate daptomycinresistance and PlySs2 lysin. These studies were conducted to evaluateand determine whether daptomycin resistance correlates with lysinresistance or sensitivity, including particularly resistance orsensitivity to PlySs2 lysin. First, daptomycin resistant clones weregenerated (with increasing MIC values), in a step-wise manner, over 21days of in vitro growth. Then, the series of daptomycin resistant cloneswere assessed with respect to lysin MIC values, by evaluating PlySs2lysin MIC. The results are depicted in FIG. 28. In the daptomycinresistant clones, daptomycin MIC rises from 1 to 18 μg/mL. PlySs2 lysinMIC decreases from 8 to between 2-4 μg/mL. These studies show thatdaptomycin resistance correlates with PlySs2 lysin sensitivity. Theseare the first studies to evaluate daptomycin resistance and lysinsensitivity. Remarkably, in these conditions resistance to daptomycinconfers increased response to lysin, providing enhanced rational forcombination or serial administration and therapy.

Example 10

Serial passage resistance studies were undertaken to assess the abilityof PlySs2 to suppress the emergence of antibiotic resistance when usedin combination with various standard-of-care antibiotics used to treatStaphylococcus aureus infections. Methods used to perform single-agentand combination serial passage experiments are described in Palmer et al(Palmer et al (2011) Antimicrobial Agents and Chemotherapy 55:3345-56)and Berti et al (Berti et al (2012) Antimicrobial Agents andChemotherapy 56:5046-53), respectively. Increases in the MIC values forthe antibiotic were assessed in triplicate for a MRSA Staphylococcusaureus strain (MW2) grown either in the presence of antibiotic alone orin the presence of antibiotic plus sub-MIC values of PlySs2. The MIC forPlySs2 for strain MW2 is 32 ug/ml (without DTT). Thus, sub-MIC values of4 ug/ml (corresponding to ⅛ MIC) or 8 ug/ml (corresponding to ¼ MIC)were chosen as the concentration of PlySs2 for these experiments. Bothdaptomycin and vancomycin were tested as exemplary antibiotics in thisstudy.

For the daptomycin experiments, it was found that over the course of the30-day study, daptomycin resistance increased significantly for allthree daptomycin-only cultures (FIG. 30). In these cultures thedaptomycin MIC values increased from the starting value of 1 ug/ml, tothe three ending values of 128, 128 and 64 ug/ml—a 64 to 128-foldincrease. For cultures that were passaged in the presence of sub-MICamounts of PlySs2 (4 ug/ml) plus daptomycin, the daptomycin MIC valuesat the end of the 30 serial passage experiment were significantly lower−4, 4, and 4 ug/ml (a 4 fold increase). Therefore, sub-MICconcentrations of PlySs2 suppressed the ability of the MRSA strain tomount daptomycin resistance by 8 to 16 fold relative to the daptomycinalone conditions. Resistance to daptomycin increased by only about 4fold in the presence of PlySs2 lysin.

For the vancomycin experiments, it was found that over the course of the25-day study, vancomycin resistance increased significantly for allthree vancomycin-only cultures. In these cultures the vancomycin MICvalues increased from the starting value of 1 ug/ml, to the three endingvalues of 16, 16 and 16 ug/ml (a 16-fold increase). For cultures thatwere passaged in the presence of sub-MIC amounts of PlySs2 (8 ug/ml)plus vancomycin, the vancomycin MIC values at the end of the 25 dayserial passage experiment were significantly lower −4, 4, and 2 ug/ml (a2 to 4 fold increase). Therefore, sub-MIC concentrations of PlySs2suppressed the ability of the MRSA strain to mount vancomycin resistanceby 4 to 8 fold relative to the vancomycin alone conditions. Resistanceto daptomycin increased by only about 4 fold in the presence of PlySs2lysin.

REFERENCES

-   1. Klevens, R. M., et al. Invasive Methicillin-Resistant    Staphylococcus aureus Infections in the United States. JAMA 298,    1763-1771 (2007).-   2. Brink, A. J. Does resistance in severe infections caused by    methicillin-resistant Staphylococcus aureus give you the ‘creeps’?    Current opinion in critical care 18, 451-459 (2012).-   3. Ben-David, D., Novikov, I. & Mermel, L. A. Are there differences    in hospital cost between patients with nosocomial    methicillin-resistant Staphylococcus aureus bloodstream infection    and those with methicillin-susceptible S. aureus bloodstream    infection? Infection control and hospital epidemiology: the official    journal of the Society of Hospital Epidemiologists of America 30,    453-460 (2009).-   4. Fischetti, V. A. Bacteriophage lysins as effective    antibacterials. Current opinion in microbiology 11, 393-400 (2008).-   5. Fenton, M., Ross, P., McAuliffe, O., O'Mahony, J. & Coffey, A.    Recombinant bacteriophage lysins as antibacterials. Bioengineered    Bugs 1, 9-16 (2010).-   6. Nelson, D., Loomis, L. & Fischetti, V. A. Prevention and    elimination of upper respiratory colonization of mice by group A    streptococci by using a bacteriophage lytic enzyme. Proceedings of    the National Academy of Sciences of the United States of America 98,    4107-4112 (2001).-   7. Witzenrath, M., et al. Systemic use of the endolysin Cpl-1    rescues mice with fatal pneumococcal pneumonia. Critical care    medicine 37, 642-649 (2009).-   8. McCullers, J. A., Karlstrom, A., Iverson, A. R., Loeffler, J. M.    & Fischetti, V. A. Novel Strategy to Prevent Otitis Media Cauesed by    Colonizing Streptococcus pneumoniae. PLOS pathogens 3, 0001-0003    (2007).-   9. Pastagia, M., et al. A novel chimeric lysin shows superiority to    mupirocin for skin decolonization of methicillin-resistant and    -sensitive Staphylococcus aureus strains. Antimicrobial agents and    chemotherapy 55, 738-744 (2011).-   10. Loeffler, J. M., Djurkovic, S. & Fischetti, V. A. Phage Lytic    Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia.    Infection and Immunity 71, 6199-6204 (2003).-   11. Entenza, J. M., Loeffler, J. M., Grandgirard, D.,    Fischetti, V. A. & Moreillon, P. Therapeutic effects of    bacteriophage Cpl-1 lysin against Streptococcus pneumoniae    endocarditis in rats. Antimicrobial agents and chemotherapy 49,    4789-4792 (2005).-   12. Grandgirard, D., Loeffler, J. M., Fischetti, V. A. & Leib, S. L.    Phage lytic enzyme Cpl-1 for antibacterial therapy in experimental    pneumococcal meningitis. The Journal of infectious diseases 197,    1519-1522 (2008).-   13. Blaser, M. Stop killing beneficial bacteria. Nature 476, 393-394    (2011).-   14. Willing, B. P., Russell, S. L. & Finlay, B. B. Shifting the    balance: antibiotic effects on host-microbiota mutualism. Nature    reviews. Microbiology 9, 233-243 (2011).-   15. Gilmer, D. B., Schmitz, J. E., Euler, C. & Fischetti, V. A.    Novel Bacteriophage Lysin with Broad Lytic Activity Protects against    Mixed Infection by Methicillin-Resistant Staphylococcus aureus and    Streptococcus pyogenes TBD (2012).-   16. Schuch, R., Fischetti, V. A. & Nelson, D. C. A Genetic Screen to    Identify Bacteriophage Lysins. in Bacteriophages: Methods and    Protocols, Volume 2: Molecular and Applied Aspects, Vol. 502 307-319    (2009).-   17. Bateman, A. & Rawlings, N. D. The CHAP domain: a large family of    amidases including GSP amidase and peptidoglycan hydrolases. Trends    Biochem Sci 28, 234-237 (2003).-   18. Whisstock, J. C. & Lesk, A. M. SH3 domains in prokaryotes.    Trends in Biochemical Sciences 24, 132-133 (1999).-   19. Rossi, P., et al. Structural elucidation of the Cys-His-Glu-Asn    proteolytic relay in the secreted CHAP domain enzyme from the human    pathogen Staphylococcus saprophyticus. Proteins 74, 515-519 (2009).-   20. Mueller, M., de la Pena, A. & Derendorf, H. Issues in    Pharmacokinetics and Pharmacodynamics of Anti-Infective Agents: Kill    Curves versus MIC. Antimicrobial agents and chemotherapy 48, 369-377    (2004).-   21. Methods for dilution antimicrobial susceptibility tests for    bacteria that grow aerobically. Vol. 32 (Wayne (Pa.): Clinical and    Laboratory Standards Institute (US), 2012).-   22. Friedman, L., Alder, J. D. & Silverman, J. A. Genetic changes    that correlate with reduced susceptibility to daptomycin in    Staphylococcus aureus. Antimicrobial agents and chemotherapy 50,    2137-2145 (2006).-   23. Donlan, R. M. & Costerton, J. W. Biofilms: Survival Mechanisms    of Clinically Relevant Microorganisms. Clinical Microbiology Reviews    15, 167-193 (2002).-   24. Cottarel, G. & Wierzbowski, J. Combination drugs, an emerging    option for antibacterial therapy. Trends in biotechnology 25,    547-555 (2007).-   25. Tallarida, R. J. Revisiting the isobole and related quantitative    methods for assessing drug synergism. The Journal of pharmacology    and experimental therapeutics 342, 2-8 (2012).-   26. LaPlante, K. L., Leonard, S. N., Andes, D. R., Craig, W. A. &    Rybak, M. J. Activities of clindamycin, daptomycin, doxycycline,    linezolid, trimethoprim-sulfamethoxazole, and vancomycin against    community-associated methicillin-resistant Staphylococcus aureus    with inducible clindamycin resistance in murine thigh infection and    in vitro pharmacodynamic models. Antimicrobial agents and    chemotherapy 52, 2156-2162 (2008).-   27. Crandon, J. L., Kuti, J. L. & Nicolau, D. P. Comparative    efficacies of human simulated exposures of telavancin and vancomycin    against methicillin-resistant Staphylococcus aureus with a range of    vancomycin MICs in a murine pneumonia model. Antimicrobial agents    and chemotherapy 54, 5115-5119 (2010).-   28. Abad, C. L., Kumar, A. & Safdar, N. Antimicrobial therapy of    sepsis and septic shock—when are two drugs better than one? Critical    care clinics 27, el-27 (2011).-   29. Fischbach, M. A. Combination therapies for combating    antimicrobial resistance. Current opinion in microbiology 14,    519-523 (2011).-   30. Loeffler, J. M., Nelson, D. & Fischetti, V. A. Rapid killing of    Streptococcus pneumoniae with a bacteriophage cell wall hydrolase.    Science 294, 2170-2172 (2001).-   31. Costerton, J. W. Bacterial Biofilms: A Common Cause of    Persistent Infections. Science 284, 1318-1322 (1999).-   32. Kiedrowski, M. R. & Horswill, A. R. New approaches for treating    staphylococcal biofilm infections. Annals of the New York Academy of    Sciences 1241, 104-121 (2011).-   33. Domenech, M., Garcia, E. & Moscoso, M. In vitro destruction of    Streptococcus pneumoniae biofilms with bacterial and phage    peptidoglycan hydrolases. Antimicrobial agents and chemotherapy 55,    4144-4148 (2011).-   34. Meng, X., et al. Application of a bacteriophage lysin to disrupt    biofilms formed by the animal pathogen Streptococcus suis. Applied    and environmental microbiology 77, 8272-8279 (2011).-   35. Schuch, R., Nelson, D. & Fischetti, V. A bacteriolytic agent    that detects and kills Bacillus anthracis. Nature 418, 884-889    (2002).-   36. Fischetti, V. A., Nelson, D. & Schuch, R. Reinventing phage    therapy: are the parts greater than the sum? Nature Biotechnology    24, 1508-1511 (2006).-   37. Manoharadas, S., Witte, A. & Blasi, U. Antimicrobial activity of    a chimeric enzybiotic towards Staphylococcus aureus. Journal of    biotechnology 139, 118-123 (2009).-   38. Rashel, M., et al. Efficient elimination of multidrug-resistant    Staphylococcus aureus by cloned lysin derived from bacteriophage phi    MR11. The Journal of infectious diseases 196, 1237-1247 (2007).-   39. Daniel, A., et al. Synergism between a novel chimeric lysin and    oxacillin protects against infection by methicillin-resistant    Staphylococcus aureus. Antimicrobial agents and chemotherapy 54,    1603-1612 (2010).-   40. Kokai-Kun, J. F., Chanturiya, T. & Mond, J. J. Lysostaphin as a    treatment for systemic Staphylococcus aureus infection in a mouse    model. The Journal of antimicrobial chemotherapy 60, 1051-1059    (2007).-   41. Dhand, A., et al. Use of antistaphylococcal beta-lactams to    increase daptomycin activity in eradicating persistent bacteremia    due to methicillin-resistant Staphylococcus aureus: role of enhanced    daptomycin binding. Clinical infectious diseases: an official    publication of the Infectious Diseases Society of America 53,    158-163 (2011).-   42. Matias, V. R. & Beveridge, T. J. Cryo-electron microscopy of    cell division in Staphylococcus aureus reveals a mid-zone between    nascent cross walls. Molecular microbiology 64, 195-206 (2007).-   43. Kashyap, D. R., et al. Peptidoglycan recognition proteins kill    bacteria by activating protein-sensing two-component systems. Nature    medicine 17, 676-683 (2011).-   44. Moise, P. A., North, D., Steenbergen, J. N. & Sakoulas, G.    Susceptibility relationship between vancomycin and daptomycin in    Staphylococcus aureus: facts and assumptions. Lancet Infect Dis 9,    617-624 (2009).-   45. Jobson, S., Moise, P. A. & Eskandarian, R. Retrospective    observational study comparing vancomycin versus daptomycin as    initial therapy for Staphylococcus aureus infections. Clinical    therapeutics 33, 1391-1399 (2011).-   46. Schweizer, M. L., et al. Comparative effectiveness of nafcillin    or cefazolin versus vancomycin in methicillin-susceptible    Staphylococcus aureus bacteremia. BMC infectious diseases 11, 279    (2011).-   47. Bern, A. D., et al. Altering the proclivity towards daptomycin    resistance in methicillin-resistant Staphylococcus aureus using    combinations with other antibiotics. Antimicrobial agents and    chemotherapy 56, 5046-5053 (2012).-   48. Sopirala, M. M., et al. Synergy testing by Etest, microdilution    checkerboard, and time-kill methods for pan-drug-resistant    Acinetobacter baumannii. Antimicrobial agents and chemotherapy 54,    4678-4683 (2010).-   49. Methods for dilution antimicrobial susceptibility tests for    bacteria that grow aerobically. Vol. 32 (Clinical and Laboratory    Standards Institute (US), Wayne (Pa.), 2012).-   50. Clinical Microbiology Procedures Handbook 3rd Ed. Washington    D.C., (ASM Press, 2010).-   51. Pereira, P. M., Filipe, S. R., Tomasz, A. & Pinho, M. G.    Fluorescence ratio imaging microscopy shows decreased access of    vancomycin to cell wall synthetic sites in vancomycin-resistant    Staphylococcus aureus. Antimicrobial agents and chemotherapy 51,    3627-3633 (2007).-   52. Zhang, Y. I-TASSER server for protein 3D structure prediction.    BMC bioinformatics 9, 40 (2008).-   53. Pettersen, E. F., et al. UCSF Chimera—a visualization system for    exploratory research and analysis. Journal of computational    chemistry 25, 1605-1612 (2004).

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of reducing the development ofantibiotic resistance to Staphylococcus or Streptococcus bacteria in ahuman comprising: administering to the human: i) a Gram-positiveantibiotic selected from: vancomycin or a glycopeptide antibiotic of thesame class; daptomycin or a lipopeptide antibiotic of the same class;and oxacillin or a beta lactam penicillin antibiotic of the same class;and ii) a lysin effective to kill Staphylococcus and/or Streptococcusbacteria, wherein the lysin comprises the amino acid sequence providedin SEQ ID NO: 1 or a variant thereof having at least 90% identity to thepolypeptide of SEQ ID NO: 1 and effective to kill Staphylococcus and/orStreptococcus bacteria, and wherein the lysin is administered at a doseat or below the minimal inhibitory concentration (MIC) dose.
 2. Themethod of claim 1, wherein both the antibiotic and the lysin areadministered at a dose below the minimal inhibitory concentration (MIC)dose.
 3. The method of claim 1 wherein the antibiotic is administered ata dose below the minimal inhibitory concentration (MIC).
 4. The methodof claim 1 wherein the lysin comprises the amino acid sequence providedin SEQ ID NO: 1 or variants thereof having at least 95% identity to thepolypeptide of SEQ ID NO: 1 and effective to kill Staphylococcus andStreptococcus bacteria.
 5. The method of claim 1 wherein theStaphylococcus bacteria is Staphylococcus aureus.
 6. The method of claim1 wherein the antibiotic is selected from vancomycin, oxacillin anddaptomycin.
 7. The method of claim 1 wherein the lysin is administeredat a dose below the minimal inhibitory concentration (MIC) dose.
 8. Themethod of claim 1 wherein the Staphylococcus bacteria ismethicillin-resistant Staphylococcus aureus.